ELSEVIER
MICROBIOLOGY
REVIEWS
FEMS Microbiology Reviews 17 (1995) 381-399
Bacteriocin release proteins: mode of action, structure, and
biotechnological application
Fimme Jan van der Wal, Joen Luirink, Bauke Oudega *
Department of'Molecular Microbiology, IMBW, BioCentrum Amsterdam, Facult3" of'Biology, De Boelelaan 1087, I081 HV Amsterdam, The
Netherlands
Received 3 May 1995; revised 21 July 1995: accepted 9 August 1995
Abstract
The mechanism by which Gram-negative bacteria like Escherichia coli secrete bacteriocins into the culture medium is
unique and quite different from the mechanism by which other proteins are translocated across the two bacterial membranes,
namely through the known branches of the general secretory pathway. The release of bacteriocins requires the expression
and activity of a so-called bacteriocin release protein and the presence of the detergent-resistant phospholipase A in the outer
membrane. The bacteriocin release proteins are highly expressed small lipoproteins which are synthesized with a signal
peptide that remains stable and which accumulates in the cytoplasmic membrane after cleavage. The combined action of
these stable, accumulated signal peptides, the lipid-modified mature bacteriocin release proteins (BRPs) and phospholipase A
cause the release of bacteriocins. The structure and mode of action of these BRPs as well as their application in the release
of heterologous proteins by E. coli is described in this review.
Keywords: Bacteriocin release protein; Escherichia coli; Gram-negative bacteria; Heterologous protein release; Stable signal peptide;
Biotechnology
Contents
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. The cell envelope of Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Protein secretion by E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
382
382
382
2. Colicins and cloacin DFI3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Genetics, mode of action, and immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Evolutionary and ecological aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
383
383
386
3. Bacteriocin release proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Effects of BRP expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
386
386
* Corresponding author. Tel.: + 31 (20) 444 7177; Fax: + 31 (20) 444 7123: E-mail: [email protected].
0168-6445/95/$29.00 © 1995 Federation of European Microbiological Societies. All rights reserved
SSDI 01 6 8 - 6 4 4 5 ( 9 5 ) 0 0 0 2 2 - 4
382
F.J. van der Wal et al. / FEMS Microbiology Ret'iews 17 (1995) 381-399
3.2. Lipid modification, processing, and subcellular localization of BRPs . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. BRP signal peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Translocation of bacteriocins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
388
389
390
4. BRPs as tools in releasing heterologous proteins from the periplasm of E. coli . . . . . . . . . . . . . . . . . . . . . . .
4.1. Secretion of heterologous proteins by E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. BRP-mediated release of heterologous proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Considerations for optimizing BRP-mediated protein release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
391
391
392
393
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
393
1. Introduction
1.1. The cell envelope o f Escherichia coli
The cell envelope of a Gram-negative bacterium,
like Escherichia coli, is a three-layered structure,
consisting of the outer membrane, the cytoplasmic
membrane and the periplasmic space in between.
The outer membrane is an asymmetric bilayer of
which the outer leaflet consists of lipopolysaccharides (LPS) and the inner leaflet of phospholipids.
This membrane functions as a permeability barrier,
protecting the cell against, for instance, bile salts,
digestive (proteolytic and lipolytic) enzymes, antibiotics and bacteriophages. The outer membrane also
prevents periplasmic proteins from leaking out [1-3].
The outer membrane itself contains specific and
non-specific diffusion pores for the uptake of nutrients and the disposal of waste products. In addition,
high-affinity receptor proteins function in the acquisition of large molecules like vitamin B j2 and ironsiderophore complexes [4-7].
The outer membrane is covalently attached to the
peptidoglycan layer in the periplasm via the murein
lipoprotein, also referred to as Lpp [8]. The peptidoglycan layer consists of a highly cross-linked threedimensional network, containing alternating residues
of the amino sugars N-acetyl glucosamine and Nacetyl muramic acid. The peptidoglycan layer sustains the shape of the cell, and its rigidity enables the
cell to endure the osmotic pressure of the cell interior [1,9]. In addition to the peptidoglycan layer, the
periplasm contains detoxifying and scavenging enzymes, and binding proteins that facilitate active
transport [ 1,10].
The last permeability barrier of the cell is the
inner or cytoplasmic membrane. This membrane is a
lipid bilayer containing many proteins involved in
energy transduction, active transport of solutes [1113] and secretion of proteins [14,15]. Fig. 1 shows a
schematic representation of the E. coli cell envelope
structure.
1.2. Protein secretion by E. coli
Many proteins synthesized in the cytoplasm of
Gram-negative bacteria are destined to reach the
periplasm, the outer membrane or the extracellular
medium. Bacteriocins belong to a class of proteins
that are released into the extracellular environment,
and the mechanism by which this occurs is exceptional.
Targeting of proteins to and translocation across
the cytoplasmic membrane is in most cases mediated
by cleavable amino-terminal signal peptides. Signal
peptides have a positively charged amino-terminus,
followed by a hydrophobic region and a non-helical
carboxyl-terminus [ 16,17]. The basic amino-terminus
and the hydrophobic core are essential for efficient
translocation, whereas the carboxyl-terminal domain
is recognized by signal peptidases [18-20]. Several
soluble and membrane-associated components have
been identified that participate in the translocation of
precursor proteins across the cytoplasmic membrane
[21,22]. In this pathway, the cytoplasmic chaperone
SecB binds to the mature region of newly synthesized precursor proteins. The SecB precursor complex is subsequently targeted to the cytoplasmic
membrane via an interaction with SecA. SecA is an
ATPase which shuttles between the cytoplasm and
the cytoplasmic membrane. At the inner side of the
cytoplasmic membrane, SecA interacts with the
translocase complex, an integral membrane protein
F.,L can der Wal et al. / FEMS Microbiology Reciews 17 (1995) 381-399
Fig. 1. Structure of the E. coli cell envelope. Abbreviations: A,
OmpA protein; BP, periplasmic binding protein; CM, cytoplasmic
membrane; IMP, inner membrane protein; LPS, lipopolysaccharide; OM, outer membrane; PG, peptidoglycan or mucopeptide or
murein; PMP, periplasmic protein; PMS, periplasmic space; PP,
pore-forming protein.
complex consisting of SecY, SecE, SecD, SecF and
SecG, which is essential for the actual insertion of
precursor proteins into and transfer across the cytoplasmic membrane. The precursor proteins are processed by signal peptidases as they traverse the
cytoplasmic membrane [19,21], rendering a cleaved
off signal peptide and a mature protein. Recent
evidence indicates that, in addition to the Sec machinery, a signal recognition particle (SRP) exists
which is essential for efficient translocation of a
subset of proteins in E. coli [23].
Proteins destined to reach the cell surface of
Gram-negative bacteria or the extracellular medium
have to cross the two membranes of the cell envelope. E. coli naturally only secretes enterotoxins,
fimbrial subunits, hemolysin and several bacteriocins
into the culture medium. Proteins synthesized with a
cleavable amino-terminal signal peptide (enterotoxins and fimbrial subunits) are first translocated
across the cytoplasmic membrane via the Sec-dependent part of the so-called general secretory pathway
(GSP) [22]. Subsequently, the periplasmic intermediates are secreted into the extracellular medium via a
terminal branch of the GSP [22]. There are two main
groups of extracellular proteins which are secreted
via a signal peptide-independent pathway. Proteins
of the first group have a carboxyl-terminal secretion
signal (like hemolysin), whereas proteins of the second group do not contain a secretion signal (like
bacteriocins).
Enterotoxins [24] and fimbrial subunits are syn-
383
thesized with a cleavable amino-terminal signal peptide, in contrast to hemolysin and bacteriocins. In E.
coli, a specific secretion route is involved in the
assembly of polymeric proteinaceous filamentous
surface appendages (pili a n d / o r fimbriae) from subunits present in the periplasm [25-27]. Several
Gram-negative bacteria possess a terminal branch of
the GSP, which is often referred to as the main
terminal branch and which is closely related to the
pullulanase secretion system of Klebsiella species
[28-30]. Pullulanase is a lipoprotein produced by
Klebsiella species. It has been shown in E. coil that
translocation of pullulanase across the cytoplasmic
membrane depends on the Sec machinery [31]. The
terminal branch of the GSP that facilitates the secretion of puilulanase can be reconstituted in E. coli,
and it has been shown that to facilitate secretion of
pullulanase at least 14 pul genes have to be expressed [32,33].
Hemolysin and bacteriocins are synthesized without a cleavable amino-terminal signal peptide and
are not secreted by one of the terminal branches of
the GSP. Hemolysin contains a well characterized
secretion signal in its carboxyl-terminal region
[34,35]. Evidence has been obtained that hemolysin
is secreted directly from the cytoplasm into the
culture medium via a trans-envelope protein complex, consisting of the transporter proteins HlyB,
HlyD and the outer membrane protein TolC [36,37].
The transporter proteins of the hemolysin secretion
system belong to the family of the so-called ABC
transporters (ATP-Binding Cassette) [14,38].
The mechanism by which bacteriocins are secreted into the culture medium is very different from
that of other secretion systems found in E. coli. In
this review, an overview of the various aspects of
bacteriocin secretion by E. coli will be presented
and the application of the bacteriocin release system
for the biotechnological production of (heterologous)
proteins will be discussed.
2. Colicins and cloacin DF13
2.1. Genetics, mode of action, and immunity
Colicins and cloacin DF13 are plasmid-encoded
bacteriocins, capable of killing E. coli cells and
F.J. z,an der Wal et aL /FEMS Microbiology Reuiews 17 (1995) 381-399
384
closely related bacterial species [25,39]. Several distinct types of colicins have been identified. Some
colicins possess RNase activity (colicin E3, E4, E6,
and cloacin DF13), other colicins display DNase
activity (colicin E2, E7, E8, and E9), a third group
has the capability to form pores in the cytoplasmic
membrane (colicin El, A, B, Ia, Ib, K, and N), and
yet another group is capable of inhibiting the synthesis of peptidoglycan and LPS O-antigens (colicin M).
Two types of colicinogenic plasmids have been
described. Small, multicopy-number plasmids, which
are not self-transmissible, encode the group A colicins like colicin A, E l - E 9 , K, N, and cloacin D13.
Large, low-copy-number plasmids, that are transmissible, encode the related group B colicins like colicins B, Ia, Ib, and M. Numerous reviews on the
various aspects of colicinogeny have been published,
and will not be cited further in this section [39-52].
In addition to bacteriocin, colicinogenic cells synthesize an immunity protein which protects the producing cells against the homologous bacteriocin. Immunity against the nuclease-type bacteriocins is provided by binding of the immunity protein in the
cytoplasm to the region of the bacteriocin possessing
the catalytic activity. Nuclease-type bacteriocins are
secreted as an equimolar complex with their corre-
sponding immunity protein. In contrast, pore-forming bacteriocins are secreted as monomers and
their immunity protein is located in the cytoplasmic
membrane. These immunity proteins protect the producing cells from exogenous bacteriocin molecules.
The genes encoding a bacteriocin and the homologous immunity protein are located in a gene cluster.
The structural and functional organization of a number of bacteriocin gene clusters are shown in Fig. 2.
Except for colicin Ia, Ib, B, and M, colicin gene
clusters contain a third gene which codes for a
protein required for the secretion of the bacteriocins,
the so-called bacteriocin release protein (BRP, also
referred to as 'kil protein' and 'lysis protein'). As
stated above, the colicin Ia, Ib, B and M gene
clusters do not contain a BRP gene. The mechanism
by which these bacteriocins reach the extracellular
environment remains unclear. The colicin Ia and Ib
gene clusters do contain additional open reading
frames, which might be involved in export of these
bacteriocins [56]. Expression of the genes contained
in the bacteriocin gene clusters is often controlled by
a promoter which can be activated by the SOS
response, for instance upon irradiation with UV or
by exposure to mitomycin C [53]. However, the
genes encoding the immunity proteins of pore-for-
P2
1
Colicin A
[
t
]2
T~
--~
--I
C01icin El
t
t
f
PI
T1
T2
CoticinE2
t
t
t
PI
T1
T2
--~--
Colitin E3
t
P1
[
t
P'm
C[oacinDF13
t
tt
t
t
Pz
TI P3
T2
T3
'
~
,
t
TI
t
T2
Fig. 2. Structural and functional organization of a number of bacteriocin operons. Genes are indicated by boxes. The direction of
transcription is indicated. PI, (mitomycin C) inducible promoters for the synthesis of bacteriocins, bacteriocin release proteins and in some
cases their immunity proteins. P2 and P3, promoters for the expression of immunity proteins; T I, T 2 and T 3, terminators of transcription.
F.J. L'an der Wal et al. / FEMS Microbiology Rel'iews 17 (1995) 381-399
ruing colicins are transcribed from a constitutive
promoter in the opposite direction with respect to the
colicin and BRP gene. Cells producing these immunity proteins always contain a sufficient amount of
these proteins in the cytoplasmic membrane, where
these immunity proteins have to provide immunity
against a few incoming pore-forming colicins.
The mode of killing of susceptible cells by bacteriocins of the four different types has been studied
for a long time and many reviews have appeared on
this interesting subject. Here, some of the aspects of
the killing action will be mentioned only briefly. In
385
order to reach their target, bacteriocin molecules first
have to cross the outer membrane of susceptible
cells. The bacteriocins bind to high-affinity receptor
proteins in the outer membrane of susceptible cells.
These receptors often function for instance in the
uptake of vitamin Bi2 and iron-siderophore complexes. Upon binding to the proper receptor, the
bacteriocin molecules are translocated across the cell
envelope via the uptake systems which are coupled
to the various receptor proteins.
Despite their different modes of action, the various colicins and cloacin DFI 3 have the same domain
Table I
Comparison of amino acid sequences of BRP precursors
Name Signal peptide
Mature BRP a
DFI3
MKKAKAI
CQANY I RDVQGGTVAPS
El
MRKKFFVGIFAINLLVG
.............
E2
MKKITGI ILLLLAVIILSA
..............
S...TA.V..L.T.
[155]
E3
MKKITGIILLLLAVIILSA
..............
S...TA.V..L.T.
[ 1 5 5 , 156]
E4
MKKITGIILLLFAAIILAA
..............
S...TA
E5
MKKITGI ILLLLAAI
ILAA
..............
S ....
E5
MKKI TWI ILLLLAAI
ILAA
......
S ....
E6
MKKITGIILLLLAVIILAA
..............
S...TA
....
VET.
[54, 159]
E7
MKKITGIILLLLAAIILAA
..............
S...TA
.... VET.
[157, 160]
E8
MKKITGIILLLLAVIILAA
..............
S...TA.V..L.T.
E9
MKKITGI
A
MKKIIICVILLAIMLLAA
. .V.NV..TG..S.S...
N
MCGKILLILFFIMTLSA
. .V.H .... K ..........
D
~c
. . . . . V. G G G S F R E E Q L P P S S S S K L I G V A I Q
FLF I L IVSGFLLVA
ILLLLAVI
I LSAWGSKPKT
H .......
Reference
S S S ELTG IAVQ
I ......
K .......
....
[152]
I I 5 3 , 1541
VET.
[157]
A ....
L.T.
[158]
A ....
L.T.
[54]
[161]
[158]
b
IVTGVSMGSDGVGNP
RLTGLKLSKRSKDPL
[701
[162]
[163]
The dots indicate identical amino acid residues as compared to the mature p C I o D F I 3 B R P ; in the other sequences only the altered amino
acid residues are indicated.
b The colicin E 9 B R P h a s a different structure as compared to the ones above the E 9 B R P ; it seems to have a comparable signal sequence
structure but the cysteine residue at position + 1 is missing. A s a result this BRP is not lipid-modified and processed; furthermore, it is short
as compared to the other BRPs. This BRP does not function in the release of colicin E 9 .
c The sequence of the signal peptide of the colicin D B R P is not known.
Taken from [ 5 4 , 7 0 , 1 5 2 - 1 6 3 ] .
386
F.J. can der Wal et al. / FEMS Microbiology Reciews 17 (1995) 381-399
structure. The central region of the bacteriocin protein is involved in binding to specific receptor
molecules of the susceptible cells, the amino-terminal part of the bacteriocin molecule is important for
the uptake of bacteriocin molecules across the cell
envelope, whereas the carboxyl-terminal part possesses catalytic or ionophore-like activity, as well as
the binding site for the homologous immunity protein.
2.2. Evolutionary and ecological aspects
As described above, the various colicins and
cloacin DF13 show the same kind of domain structure. They also display high levels of sequence similarity in the various functional domains, suggesting
that these colicins and cloacin DFI 3 share a common
ancestor or common ancestors. The immunity proteins and the BRPs also show a high degree of
sequence similarity (see for instance Table 1 for
detailed information on the full lengths of the various BRPs; more information on these structures is
provided in Section 3.2 and Section 3.3). The organization of the various colicin gene clusters appears to
be very similar. All these findings have led to the
hypothesis that the various colicin gene clusters were
formed by recombination events. A few colicinogenic plasmids contain a gene for an additional
immunity protein. In one case, this is probably the
result of a transposition event as suggested by the
presence of a degenerate transposon-like structure
[40,54-57].
In nature, about one-third of the E. coli population is colicinogenic [43,55,58,59], which at least
suggests some evolutionary advantage of colicinogeny. Colicinogeny might protect a bacterial
population against competing bacteria and possibly
ensures the maintenance of colicin plasmids, which
often code for additional factors like fimbriae,
siderophores, and resistance to serum [40,60]. It has
been suggested that colicinogeny may only be beneficial to bacteria when they are outside their intestinal habitat, because anaerobic conditions would not
permit an efficient expression of at least some bacteriocins and furthermore, high proteolytic activity
would lead to proteolytic degradation of the bacteriocins [44]. However, tests to demonstrate the selective advantage of colicinogeny for bacteria outside
their natural habitat have never given conclusive
results, due, in part, to poor experimental design. In
contrary, studies on the molecular evolution of colicin plasmids with emphasis on the endonuclease
types of colicins [55] have provided information that
E. coli strains do have a competitive advantage
inside their own intestinal habitat, since expression
of several of these colicins is actually increased
under anaerobic conditions and proteases appeared to
be absent in the colon. The relatively high frequency
of colicin encoding plasmids in isolates of pathogenic
E. coli also supports this hypothesis [40,61,62]. It
has been shown that upon introduction of colicinogenic E. coli, non-colicinogenic E. coli cells are
displaced, indicating that colicinogeny can provide a
competitive advantage [63].
3. Bacteriocin release proteins
3.1. Effects of BRP expression
Most bacteriocins are secreted into the extracellular medium by the action of so-called bacteriocin
release proteins (BRPs). A role for BRPs in the
secretion of bacteriocins was first shown by analysis
of insertion and deletion mutants of the pCloDFI3encoded BRP gene [64-70]. The requirement for
BRP in bacteriocin release was confirmed by complementation experiments in which the expression of
the bacteriocin and its BRP were separately controlled [71,72]. With respect to the secretion of heterologous colicins, BRPs are functionally interchangeable. This was shown for the secretion of
colicin E2 by simultaneous expression of the colicin
El, E3, E7, A, or D gene clusters, and suggests a
common mode of action of the BRPs [73].
BRP-mediated secretion of bacteriocins is semispecific, because the release of bacteriocin molecules
is accompanied by the release of a subset of cytoplasmic proteins (for instance elongation factor G
and Tu, and chloramphenicol acetyltransferase) and
many periplasmic proteins, like fl-lactamase, alkaline and acid phosphatase and RNase I [15,74-79].
Full induction of a colicin gene cluster causes a
decline in culture turbidity, which is often referred to
as quasi-lysis. This decline in culture turbidity is not
caused by insufficient protection by immunity pro-
F.J. t,an der Wal et al. / FEMS Microbiology Ret'iews 17 (1995) 381-399
teins and the activity of bacteriocins at full induction
conditions, but by the activity of the BRP and its
stable signal peptide. The observed decline in culture
turbidity coincides with bacteriocin secretion and the
release of other proteins. Furthermore, upon full
induction of a colicin gene cluster, protein biosynthesis in general is inhibited and the capacity for transport of thiomethyl /3-o-galactosidase and c~-methyl
glucoside is lost [80,81]. The effects of full induction
on the decline in culture turbidity and the inhibition
of protein synthesis are demonstrated in Fig. 3.
Expression of the subcioned pCloDF13 BRP has
been shown to cause a decline in the membrane
I
i
|
I
I
i
A
0.8.
b 0.6~
0.1.
P
~
IPTG
-
IPTG
0.2.
D
o
i
20
i
40
i
60
i
80
i
100
!
120
i
i
i
i
I
i
B
-
IPTG
x
v
c 60
~ 40
o
0
~
P
J
20
T
~
40
r
60
G
,
80
i
100
ll20
Time after induction (min)
Fig. 3. Time course of the effect of the pCloDF13-derived BRP on
the culture turbidity and on the incorporation of 3H-labelled amino
acid residues. Cells of E. coli harboring a plasmid encoding the
subcloned pCloDF13-BRP were cultured in broth and fully induced [82]. At the time points indicated, the turbidity of the
cultures was measured at 660 nm (A). For labelling, samples of 1
ml were collected, supplemented with 20 /zCi of a mixture of
3H-amino acids and further incubated. After 5 rain, 10%
trichloroacetic acid was added, precipitates were collected, washed
twice with cold acetone and solubilized in 200 /J,l of SDS-PAGE
solubilization mixture. Samples of 5/zl were counted directly in a
liquid scintillation counter (B). A decrease in incorporation of
radioactivity was interpreted as a decrease in the protein biosynthesis capacity of the cells [82].
387
potential at the same time or shortly after quasi-lysis
occurs, whereas the overall capacity for protein
biosynthesis and the transport of Mg 2+ ions are
strongly affected before quasi-lysis is apparent [82].
These events were also observed when the complete
cloacin gene cluster was fully expressed [82]. It
should be stressed that the phenomena described
above, which occur as a result of full induction, are
the result of the activity of the high-level expressed
BRP a n d / o r its stable signal peptide (see below).
They do not result from a decreased immunity a n d / o r
an increased bacteriocin activity. Eventually, loss of
viability is observed on broth agar plates (lethality).
Since the BRPs cause quasi-lysis and lethality they
are also referred to as 'lysis proteins' and 'kil proteins' [39,43,52].
The effects of induction of a colicin gene cluster
on the cell resemble the effects of bacteriophage
infection or treatment with EDTA, which are known
to be caused by activation of the detergent-resistant
outer membrane phospholipase A (PIdA) of E. coli.
Electron microscopy revealed that full induction of
the pCloDFI3 gene cluster leads to 'blebs' in the
outer membrane (Oudega et al., unpublished results)
[83]. Furthermore, low-level expression of the
pColE1 BRP results in the release of various constituents of the cell envelope into the medium [84,85].
Quasi-lysis is accompanied by the release of a subset
of cytoplasmic proteins into the culture medium, and
the cells become sensitive to some externally added
proteins/enzymes, like for instance lysozyme [86].
This indicates a semi-selective increase in membrane
permeability. This permeability increase is semiselective and not the result of a general leakage,
since not all cytoplasmic proteins, like for instance
8-galactosidase, or large molecules, like DNA or
RNA, are released during quasi-lysis.
A possible role for PIdA activity in bacteriocin
release has been investigated by Pugsley and
Schwartz [86]. The activity of PIdA in E. coli cells
expressing the colicin E2 gene cluster increased 3040-fold prior to quasi-lysis and bacteriocin release,
indicating that these phenomena are the consequences of induced PIdA activity [86]. It was shown
that the major outer membrane phospholipid, phosphatidylethanolamine (PE) is degraded, and that the
amounts of free fatty acids and lysoPE, the breakdown products of PE, increased [79,86-88]. The
388
F.J. t,an der Wal et al. / FEMS Microbiology Ret'iews 17 (1995) 381-399
increased membrane permeability might be due to
the membrane perturbing properties of the accumulated lysophospholipids [89-91]. Additional evidence for the involvement of PidA in permeabilization of the cell envelope came from the observation
that strains defective in PIdA (pldA) remain impermeable to lysozyme upon expression of the colicin
E2 gene cluster [86]. Furthermore, colicinogenic pldA
strains were shown to be defective in quasi-lysis and
bacteriocin release [86-88].
The requirement for the activation of PIdA can be
bypassed by other ways of membrane permeabilization. Release of bacteriocins by pldA strains can be
achieved by treatment of the cells with the detergent
Triton X-100 [73,86,92]. Furthermore, colicin A is
released by tolQ strains, which are known to be
leaky for periplasmic proteins [93]. However, in both
cases, a functional BRP remains necessary for bacteriocin release, suggesting that BRPs cause additional
modifications of the cell envelope to provoke bacteriocin release.
Based on the fact that BRPs and some bacteriophage lysis proteins are structurally related to each
other, it has been suggested that the molecular action
of BRPs requires the E. coli autolytic system analogous to the bacteriophage lysis proteins [94]. However, it has been demonstrated that the E. coli autolytic enzymes are not required for quasi-lysis and
bacteriocin secretion [95].
3.2. Lipid modification, processing, and subcellular
localization of BRPs
BRPs are synthesized as precursors polypeptides
(preBRPs) ranging in size from 45 to 52 amino acid
residues. The nucleotide sequence of most preBRPs
has been determined. BRPs are very similar in their
primary structure (Table 1). All preBRPs contain a
Leu-X-Y-Cys sequence around the signal peptide
cleavage site, in which X and Y represent small
neutral amino acid residues at the carboxyl-terminus
of the signal peptide, whereas cysteine is the first
residue of the mature protein. This so-called lipobox
is required for lipid modification and processing of
bacterial lipoproteins by signal peptidase II (SPaselI)
[96,97].
Biogenesis of the most abundant E. coli lipoprotein, the murein iipoprotein (Lpp), consists of three
successive steps [98]. First, a diacylglyceryl moiety
is covalently attached to the sulfydryl group of the
cysteine residue of the prolipoprotein. Subsequently,
the signal peptide is cleaved off by SPaselI, which
presumably occurs at the periplasmic side of the
cytoplasmic membrane [19]. Finally, a fatty acid is
covalently attached to the free amino group of the
cysteine residue [98]. The lipoprotein nature of the
colicin A-, E2-, El-, and pCIoDF13 BRPs has been
investigated [72,88,99,101]. It was shown that these
BRPs could be labelled with radioactive glycerol or
palmitate, similar to Lpp [96], and that processing of
the BRP precursors was inhibited by globomycin, an
antibiotic which specifically inhibits SPaselI [102].
Furthermore, substitution of the cysteine residue in
the lipobox of the BRP precursor was shown to
prevent lipid modification of the BRPs.
Processing of pre-BRPs is much slower than processing of pre-Lpp. Pre-Lpp is processed within 15 s
in vivo [103], whereas processing of the colicin Aand pCloDFl3-encoded BRP precursors takes minutes for completion. This is unusually slow, also
when compared to the processing rate of other precursor molecules, and allows the detection of both
unmodified and lipid-modified radiolabelled BRP
precursor by SDS-PAGE [104,105]. In Fig. 4, the
identification of a mature BRP, its stable signal
peptide, lipid-modified precursor, and the effects of
globomycin on precursor processing are shown. Slow
processing is not characteristic for all BRPs. The
colicin El BRP is processed in seconds. The mature
colicin El BRP is a lipoprotein, and treatment with
globomycin did result in a defective accumulation of
lipid-modified mature form of the colicin El BRP.
Thus globomycin appears to affect the processing
and lipid-modification of the colicin El BRP, although the unprocessed precursor of this BRP was
not found for unknown reasons [100].
The colicin El- and pCloDFI3 BRP strongly
depend on SecA and SecY for their translocation
across the cytoplasmic membrane [106,107], as does
Lpp [108,109]. However, the colicin A BRP appeared to be less dependent on SecA and SecY, since
this BRP was found to be slowly processed in secA
and secY mutants [106]. Furthermore, it was shown
that transiocation of the pCIoDFI3 BRP depends on
SecB [107], in contrast to Lpp [108,109]. The high
similarity of the primary structures of the mature
F.J. uan der Wal et al. / FEMS Microbiology Reriews 17 (1995) 381-399
plasmid:
plNIllA!
globomycin:
+
pJL22(Sphl)
+
43.0 29.0 -
18.4 -
14.3 -
6.3
i
3.4
-
2.3-
1
2
3
4
Fig. 4. Identification of plasmid-encoded gene products by tricine
SDS-PAGE and fluorography. Cells of E. coli FTP4170 harboring either plNIIIAI (lanes 1 and 2) or pJL22 (SphI) (lanes 3 and
4) were labelled and analysed [72]. Plasmid plNIIIAI is an empty
vector and was used as control. Plasmid pJL22 (SphI) is the
vector plus the subcloned BRP gene of pCIoDF13. The presence
or absence of globomycin during labelling is indicated above the
figure by a plus or a minus. The position of marker polypeptides
is indicated at the left of the figure (kDa). Symbols: r-i, lipid-modified BRP p r e c u r s o r ; . , mature BRP; * , stable BRP signal
peptide; 0 , mature lipoprotein Lpp.
portions of the colicin El- and pCloDF13 BRPs (see
Table 1) might be the cause of the shared requirement for SecA and SecY, but the molecular basis for
the differences in SecB dependency between the
various lipoproteins remains to be determined.
BRPs are predominantly located in the outer
membrane of the cell envelope [72,88,93,99,104,
110-113]. It has been shown that lipid modification
of the BRPs is important for their localization. A
pCIoDF13 BRP in which the cysteine residue in the
lipobox is replaced by another residue is mainly
located in the cytoplasmic membrane [72]. Lipid
389
modification of the BRPs is also important for their
functioning, since unmodified mutant BRPs do not
function in quasi-lysis and bacteriocin release
[72,88,99,114]. In addition, it has been shown that an
unmodified mutant precursor of the colicin A BRP,
which was processed via an alternative pathway by
SPaseI, does not function in quasi-lysis and bacteriocin release [88]. Taken together, these observations
indicate that lipid modification of BRPs is important
for their localization and functioning. However, lipid
modification is not the only feature to determine the
final location of lipoproteins [115]. The localization
of a lipoprotein has been shown to shift from the
outer membrane to the cytoplasmic membrane upon
substitution of the Ser residue at position + 2 of the
mature protein by the negatively charged residue
Asp residue, and vice versa [116]. However, a hybrid
BRP-derived protein containing an Asp residue at
position + 2 of the mature protein remained predominantly located in the outer membrane [112]. This
suggests that a different part of the mature BRP
contains information for its outer membrane localization.
3.3. BRP signal peptides
An unusual feature of most BRP signal peptides is
that they are not proteolytically degraded after processing of the BRP precursor by signal peptidase II.
Other signal peptides, like that of Lpp, are rapidly
degraded after cleavage from the precursor [117] by
signal peptide peptidases. Stability of the signal peptides of the colicin A-, E2-, and pCIoDFI3 BRPs
was determined by pulse-chase labelling experiments [88,99,101]. In contrast, the colicin El BRP
signal peptide appears to be unstable [100], whereas
the possible stability of other BRP signal peptides
has not been investigated. Stability appears to be an
intrinsic property of most BRP signal peptides, since
the colicin E2- and pCIoDF13 BRP signal peptides
can be visualized on a gel after radiolabelling, when
they are expressed as separate entities [111,114]. By
constructing hybrid signal peptides consisting of a
part of the unstable Lpp signal peptide and a part of
the stable pCIoDFI3 BRP signal peptide, it was
shown that the amino-terminal segment of the BRP
signal peptide together with the carboxyl-terminal
alanine residue are important for stability [118].
390
F.J. van der Wal et al. / F E M S Microbiology Reviews 17 (1995) 381-399
The cleaved off stable colicin A- and pCloDFl3
BRP signal peptides accumulate exclusively in the
cytoplasmic membrane [93,111]. It has been shown
that quasi-lysis and lethality are partly caused by the
stable signal peptides, by expressing the colicin E2and pCloDF13 BRP signal peptides as separate entities [111,114]. The molecular mechanism by which a
stable signal peptide causes quasi-lysis and lethality
is not clear. In vitro experiments suggested that
accumulation of synthetic signal peptides causes
jamming of the export machinery in inverted cytoplasmic membrane vesicles [119]. However, expression of the pCloDF13 BRP does not result in the
accumulation of precursor proteins [82]. Previously,
it was demonstrated that protein biosynthesis is inhibited upon expression of the colicin E1 BRP [80].
More recently, it was shown that both the stable
pCloDF13 BRP signal peptide, expressed as a separate entity, and the complete pCloDFl 3 BRP strongly
inhibit protein biosynthesis and affect the transport
of Mg 2+ ions before quasi-lysis occurs [82]. This
indicates that the stable BRP signal peptides are
partly responsible for quasi-lysis and lethality.
To investigate a possible role of the stable signal
peptide in bacteriocin release, the stable pCloDF13
BRP signal peptide was replaced by the unstable Lpp
signal peptide [105]. The resulting hybrid Lpp-BRP
is correctly targeted to the outer membrane [111,112]
and still causes quasi-lysis, lethality and leakage of
periplasmic proteins. However, this Lpp-BRP is not
capable of provoking the release of cloacin DF13
anymore, suggesting that the stable pCloDF13 BRP
signal peptide is involved in bacteriocin release [ 105].
Studies with hybrid signal peptides consisting of a
part of the unstable Lpp signal peptide and a part of
the stable pCloDF13 BRP signal peptide suggested a
correlation between signal peptide stability and bacteriocin release [llS]. Furthermore, the stable
pCloDF13 BRP signal peptide expressed as a separate entity is unable to provoke the translocation of
cloacin DFl3 across the cytoplasmic membrane
[lll], but when it is expressed in trans with the
Lpp-BRP it can complement the Lpp-BRP for functioning in the release of cloacin DF13 [151]. Taken
together, these observations strongly suggest that, in
addition to the mature lipid-modified BRP, the stable
signal peptide is required for efficient bacteriocin
release.
There seems to be a discrepancy between the
requirement for signal peptide stability in the release
of colicin E1 and cloacin DF13. Possibly, the colicin
E1 BRP signal peptide is of intermediate stability,
analogous to a hybrid signal peptide consisting of the
10 amino-terminal residues of the Lpp signal peptide
and the 10 carboxyl-terminal residues of the
pCloDFI3 BRP signal peptide. This hybrid signal
peptide (10Lppl0BRP) functions in the release of
cloacin DF13, albeit with reduced efficiency [118].
Similar to the colicin E1 BRP signal peptide, this
hybrid signal peptide does not accumulate upon processing of the BRP precursor. However, when expressed as a separate entity, the hybrid signal peptide
can be detected by SDS-PAGE at the start of a
pulse-chase labelling experiment, and remains detectable for at least l0 rain. It is conceivable that the
lack of stable signal peptide is compensated for by
the relatively rapid processing of the colicin E1 BRP
precursor [100], which might allow the temporary
accumulation of the cleaved off signal peptide.
3.4. Translocation of bacteriocins
E. coli strains harboring Col plasmids or the
pCIoDF13 vector can secrete the encoded bacteriocins into the culture medium, which is accompanied
by the release of a subset of host proteins [39,43,120].
It has been suggested that the stable signal peptide,
the mature BRP and PldA cooperate in the formation
of trans-envelope pores for bacteriocin release [105].
The fact that colicin A and cloacin DF13 do accumulate in the cytoplasm of pldA strains, and not in the
periplasm, is in accordance with this hypothesis ([88];
Luirink and Oudega, unpublished results). Moreover,
bacteriocins accumulate in the cytoplasm when the
BRP is not processed [88], or when the stable signal
peptide is expressed without the mature BRP [l 11].
The release of periplasmic proteins concomitant with
bacteriocin release might be explained by the activation of PldA in regions of the outer membrane that
are not part of the trans-envelope pores.
Attempts to visualize these putative trans-envelope pores by electron microscopy have not been
successful (Oudega et al., unpublished results); however, some indirect evidence for their existence has
been obtained. Membranes of BRP-producing cells
F.J. van der Wal et a l . / FEMS Microbiology Ret'iews 17 (1995) 381-399
are difficult to separate by isopycnic sucrose density
gradient centrifugation experiments, and proteins of
the two types of membranes are detected in fractions
of intermediate density [93,113] (Stegehuis and
Oudega, unpublished results). This suggests that upon
expression of a BRP the density of the various
membrane fractions is changed. Membranes of pldA
strains producing the colicin A BRP are also difficult
to separate, indicating that BRPs cause alterations in
the cell envelope independent of PldA [93]. As discussed above, neither BRP nor PIdA alone are able
to provoke the release of bacteriocin, which supports
the idea that the simultaneous effects of the BRP and
PIdA are required for the formation of the putative
trans-envelope pores. The stable signal peptides
might contribute to the formation of trans-envelope
pores at the level of the cytoplasmic membrane. In
vitro experiments showed that membrane insertion of
the signal peptides of PhoE and the M 13 phage coat
protein results in a loss of bilayer structure [ 121,122].
In these experiments, artificial bilayer systems were
used which lacked signal peptide peptidases, and
therefore possibly resemble the situation in which a
stable BRP signal peptide inserts into the E. coli
cytoplasmic membrane. Taken together, the stable
signal peptide and the mature BRP cause damage to
the cytoplasmic and to the outer membranes upon
which PldA is activated, possibly by the membrane
perturbing activity of the BRP, analogous to PldA
activation by the membrane perturbants polymyxin B
and mellitin [123].
4. BRPs as tools in releasing heterologous proteins
from the periplasm of E. coli
4.1. Secretion of heterologous proteins by E. coli
Although E. coli is not very proficient in secreting proteins into the culture medium, its accessibility
to genetic manipulations and the vast amount of
knowledge on its physiology have led to several
investigations concerning the use and adaptation of
the E. coli secretion systems for other proteins.
Secretion of proteins of interest would be advantageous for several reasons. Firstly, proteolysis of
secreted proteins is expected to be limited [ 124,125].
391
For example, expression of recombinant proteins
(hemolysin fusions) in cells containing the transport
proteins HlyB and HlyD results in the secretion of
these recombinant proteins, whereas they are degraded in the cytoplasm of cells lacking these transport proteins [126]. Secondly, since Gram-negative
bacteria secrete a limited number of proteins, purification of proteins from the medium is relatively easy
owing to little contamination with proteins of the
host [ 19,124]. Thirdly, formation of disulfide bridges
is promoted when heterologous proteins are released
from the reducing cytoplasmic environment into the
periplasm or into the extracellular medium [127132]. Fourthly, secretion of proteins might help preventing the formation of intracellular protein aggregates upon high-level expression. Finally, the biological activity of some eukaryotic proteins is affected
by the presence of an amino-terminal methionine
residue which is circumvented by producing such
proteins in E. coli as precursors with a cleavable
amino-terminal signal peptide [ 124,133].
A lot of effort has been put into the adaptation of
the E. coli hemolysin and bacteriocin secretion systems for the secretion of heterologous proteins into
the medium. Early studies have shown that E. coli
cells expressing the accessory membrane proteins
HlyB and HlyD can secrete a chimerical LacZ-OmpF
protein into the culture medium when it is fused to
the carboxyl-terminal region of hemolysin [134]. The
carboxyl-terminal region of hemolysin contains a
well characterized secretion signal [34,135] and has
been applied to guide various proteins into the extracellular medium. Using this system, secretion of the
variable domains of an antibody [126] and of the E.
coli cytoplasmic proteins chloramphenicol acetyltransferase and fl-galactosidase [135] has been
achieved. In addition, the mammalian protein
prochymosin was found extracellularly when fused
to the hemolysin secretion signal [135]. This is interesting because it has been shown that signal peptide
directed translocation of prochymosin across the cytoplasmic membrane is not possible in E. coli [136].
Attempts to apply the bacteriocin secretion system
for the release of proteins fused to bacteriocins or
parts of bacteriocin polypeptides have not been successful [ 137-140], possibly because bacteriocins do
not possess secretion signals [78]. Nevertheless, the
observation that BRPs can promote the leakage of
392
F.J. t~'ander Wal et al. / FEMS Microbiology Reeiews 17 (1995) 381-399
periplasmic proteins has led to the use o f s u b c l o n e d
BRPs in achieving the release of heterologous proteins into the culture m e d i u m from the E. coli
periplasm (see below) and in one case from the
cytoplasm [141]. As an alternative for BRPs, iysis
proteins o f bacteriophages have been used to release
proteins from the E. coli periplasm [142,143].
4.2. B R P - m e d i a t e d r e l e a s e o f h e t e r o l o g o u s p r o t e i n s
Proteins o f different origin and size have been
released into the culture m e d i u m by s i m u l t a n e o u s l y
expressing the target protein and a BRP. In most
cases a cleavable a m i n o - t e r m i n a l signal peptide was
used to direct the target protein into the periplasm
(Table 2). Several proteins of prokaryotic and h u m a n
origin have been released by using a o n e - p l a s m i d
system e n c o d i n g both the colicin E l B R P and the
target protein. In this system, the colicin E l B R P is
constitutively expressed at a low level, whereas the
target protein is expressed from a B a c i l l u s penicillinase promoter, and is directed to the periplasm by its
natural signal peptide or the B a c i l l u s penicillinase
signal peptide. To optimize B R P - m e d i a t e d protein
release, several expression vectors have been constructed, e n c o d i n g a BRP-controlled by an inducible
promoter. In a recent study, the p C l o D F 1 3 BRP and
a target protein, the E. coli periplasmic molecular
chaperone FaeE, were placed under control of separately inducible promoters. In addition, in the latter
study it has been shown that the efficiency o f BRPmediated protein release increased when a oneplasmid system is used instead of a b i n a r y vector
system [151].
As m e n t i o n e d before, secretion of proteins is expected to have several advantages. For example,
h u m a n calcitonin (hCT) expressed as a fusion to
penicillinase was found extracellularly, whereas cy-
Table 2
Proteins released from the E. coli periplasm by the activity of a BRP
Target protein
Size (kDa)
Signal peptide a
Type of BRP
Reference i
Bacillus penicillinase
Aeromonas xylanase L
Bacillus N-4 cellulase
Bacillus 1139 cellulase
25
135
58
92
21
29
37
27
17
29
55
29
21
40
29
55
25
Homologous
Homologous
Homologous
Homologous
Penicillinase
Penicillinase
Penicillinase
Penicillinase
Penicillinase
Homologous
Homologous
Homologous
OmpA
OmpA
Homologous
Homologous
Homologous
ColEI b
ColE1 b
ColE1 b
ColE1 b
ColE 1 b
ColE 1 b
ColEl b
ColE 1 b
ColEI b
ColEI d.e
ColEI 0x
ColEI r
CloDF13 c
CloDFI 3 ~
CloDF13 g
CIoDF 13 g
CIoDF13 ~
[147,164,165]
[166]
[166]
[166]
[ 167,165] c
[ 128,168]
[ 169]
[ 170]
[171]
[172]
[172]
[149]
[ 144]
[ 151]
[I 73] h
[ 150,173] h
[151]
Human growth hormone d
Human IgG Fc region
Human chimeric IgE/IgG Fc d
Human calcitonin
Human tumor necrosis factor-a d
fl-Lactamase
Bacillus a-amylase
fl-Lactamase
Human growth hormone ~
Guar a-galactosidase e
/3-Lactamase
Bacillus a-amylase
FaeE e
Signal peptide used for targeting of the protein, designated 'homologous' when the natural signal peptide of the target protein was used.
b ColEI-BRP is constitutively expressed at a low level in a one-plasmid system which also encodes the target protein.
Expression from single, double, or triple penicillinase promoters.
d Chemically synthesized gene.
e Gene expression under control of an IPTG-inducible promoter.
f Gene expression under control of a temperature sensitive promoter.
g Gene expression under control of the mitomycin C-inducible SOS promoter.
h Experiments were carried out in a small bioreactor, a cell recycle system, or a 20-1 fermenter.
F.J. uan der Wal et al. / FEMS Microbiology Reciews 17 (1995) 381-399
toplasmic hCT was suggested to be degraded. Furthermore, a-galactosidase has been shown to be
degraded in the cytoplasm, but not in the periplasm
or in the extracellular medium [151]. Proteins released by the action of a BRP can easily be isolated
from the culture medium, as has been shown for
human growth hormone (hGH), released by concomitant expression of the pCloDF13 BRP [144].
Furthermore, disulfide bridges in hGH released by
the action of the colicin El BRP have been shown to
be formed correctly, and the protein showed equal
biological activity as compared to authentic hGH.
Correct formation of disulfide bridges has also been
shown for the recombinant human IgG-Fc. This protein was released as a 55-60-kDa dimer with correct
interchain as well as intrachain disulfide bridges.
Furthermore, human tumor necrosis factor-a was
released as a dimer or a trimer, and the biological
activity of the released form was higher than that of
the cytoplasmic protein, possibly due to differences
in the tertiary structure. In addition, the E. coli
periplasmic molecular chaperone FaeE was suggested to be released as a dimer, which is its natural
form in the periplasm [145].
Secretion of proteins might prevent the formation
of inclusion bodies. For instance, overproduction of
hGH has been shown to result in the formation of
protein aggregates [141,146], which can be prevented
by simultaneously expressing a BRP. In addition to
the colicin El- and pCloDF13 BRP, the colicin A
BRP was also used to obtain extracellular correctly
folded and biological active hGH [141]. In this study,
the hormone peptide, amino-terminally extended with
a methionine residue (Met-hGH), was directly released from the cytoplasm. The amount of accumulated Met-hGH was about 20 times higher than that
of mature hGH, targeted by its natural signal peptide
to the periplasm, since the precursor was slowly
processed and appeared to be sensitive to proteolysis
in the cytoplasm. This also shows that efficient
secretion vectors are indispensable for optimal protein release by a BRP.
Other studies, directed towards the applicability
of BRPs on a semi-large scale [147,148], in a 20-1
fermentor [149] and in a cell recycle system [150],
have suggested that it is feasible to use BRPs in
continuous cultures in order to isolate large amounts
of a protein of interest.
393
4.3. Considerations .for optimizing BRP-mediated
protein release
BRPs have been shown to be applicable in mediating the release of a wide variety of heterologous
proteins, ranging in size from about 20 to over 100
kDa. Since expression of a BRP causes the release of
almost the entire periplasmic content [149], expression and processing of the target protein should be as
efficient as possible to selectively enrich the culture
medium with the target protein. Efficient inducible
expression/secretion vectors are indispensable, and
preferably a one-plasmid system should be used to
simultaneously express the target protein and the
BRP. Other improvements can be made by using
protease-deficient strains and by optimizing cultivation conditions [ 147,151 ].
High-level expression of BRPs causes quasi-lysis
and lethality. These phenomena are in part caused by
accumulation of the stable cleaved off BRP signal
peptides in the cytoplasmic membrane, and in part
by the mature lipid-modified BRPs. The use of the
colicin E1 BRP has the advantage that this BRP is
targeted by an unstable signal peptide, whereas the
pCloDFI3- and colicin A BRPs are targeted by
stable signal peptides. The pCIoDFI 3 BRP has been
optimized for protein secretion by using the unstable
Lpp signal peptide for targeting [151 ]. Furthermore,
preliminary results showed that mutant Lpp-BRPs
can be constructed which do not cause quasi-lysis
and lethality anymore, and that these mutated BRPs
are still capable of inducing the release of fl-lactamase from the periplasm [151]. These mutant
pCloDFI 3 BRPs have amino acid substitutions in the
central region of the mature BRP. The use of such
mutated BRPs will certainly improve the BRP-mediated release of heterologous proteins from the E. coli
periplasm into the culture medium.
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