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International Journal of Medical Microbiology

journal homepage: www.elsevier.com/locate/ijmm

Mini Review

Import and export of bacterial protein toxins

Volkmar Braun ∗ , Stephanie Helbig, Silke I. Patzer, Avijit Pramanik, Christin Römer
Max Planck Institute for Developmental Biology, Department of Protein Evolution, Spemannstrasse 35, 72076 Tübingen, Germany

a r t i c l e i n f o a b s t r a c t

Keywords: The paper provides a short overview of three investigated bacterial protein toxins, colicin M (Cma) of
Colicin M Escherichia coli, pesticin (Pst) of Yersinia pestis and hemolysin (ShlAB) of Serratia marcescens. Cma and Pst
Pesticin are exceptional among colicins in that they kill bacteria by degrading the murein (peptidoglycan). Both
Hemolysin
are released into the medium and bind to specific receptor proteins in the outer membrane of sensitive
Import
E. coli cells. Subsequently they are translocated into the periplasm by an energy-consuming process using
Export
Activity the proton motive force. For transmembrane translocation the colicins unfold and refold in the periplasm.
In the case of Cma the FkpA peptidyl prolyl cis-trans isomerase/chaperone is required. ShlA is secreted
and activated through ShlB in the outer membrane by a type Vb secretion mechanism.
© 2014 Elsevier GmbH. All rights reserved.

Introduction murein precursor resulting in undecaprenol and 1-pyrophospho-

MurNAc-(pentapeptide)-GlcNAc (Harkness and Braun, 1989; El
Bacteria secrete selected proteins by diverse, yet distinct mech- Ghachi et al., 2006). These degradation products cannot be used
anisms. For gram-negative bacteria at least seven protein secretion anymore for murein biosynthesis. Pst cleaves the glycan chain of
types and various subtypes are known. In addition, proteins exist murein between C1 of MurNAc and C4 of GlcNAc (lysozyme activ-
which are secreted without a specific secretion mechanism. These ity; Vollmer et al., 1997). Plasmid-encoded Cma is made by E. coli
are colicins which are released by nearly half of Escherichia coli nat- and kills E. coli cells. Plasmid-encoded Pst is made by Yersinia pestis
ural isolates and kill competing sensitive cells. Despite co-synthesis and kills Yersinia strains. It displays all characteristics of colicins
of immunity proteins which confer resistance to the cognate col- and is, therefore, listed here as a colicin.
icins, a small proportion of cells in a population lyses when colicins Hemolysin from S. marcescens has been included in our inves-
are overproduced under stress conditions. In contrast to unspe- tigation (Braun et al., 1987) because it was at that time one of the
cific export highly specific and sophisticated colicin import systems few proteins that were secreted by Enterobacteriaceae and had not
exist in sensitive cells. According to their mode of action colicins been investigated. The S. marcescens hemolysin structure and secre-
enter the periplasm, the cytoplasmic membrane, or the cytoplasm tion mechanism turned out to be completely different from the
of sensitive cells. For all colicins import starts with binding to spe- well-studied E. coli hemolysin.
cific receptor proteins exposed at the cell surface. Sensitivity of cells
is largely determined by those receptors (Braun et al., 2002; Jakes Domain structure of the E. coli colicin Cma
and Cramer, 2012).
Here, two colicins from E. coli and Y. pestis and the hemolysin of Cma consists of three domains which are characteristic of col-
S. marcescens were investigated by us and will be discussed with icins. The central domain serves to bind Cma to the outer membrane
respect to their cellular export and import. Colicin M (Cma) and receptor protein FhuA that concomitantly serves as a receptor
pesticin (Pst) were studied because only preliminary data existed for several phages and as a transporter for the iron chelator fer-
and they differ from other colicins in that they primarily cause richrome as well as for antibiotics derived from ferrichrome. The
lysis of cells. All other colicins either degrade DNA or RNA in the N-terminal domain is required for translocation across the outer
cytoplasm or form pores in the cytoplasmic membrane resulting membrane into the periplasm, and the C-terminal domain encodes
in collapse of the membrane potential and later cell lysis. Cma the phosphatase activity (Fig. 1). Deletion of the hydrophobic ␣1
inhibits murein biosynthesis by cleavage of the ester bond in the helix close to the N-terminus (Fig. 1; gray) abolishes binding of
Cma to FhuA. However, phosphatase activity is retained as demon-
strated in an assay that bypasses the FhuA function (Helbig and
∗ Corresponding author. Braun, 2011). Mutations in ␣1 strongly reduce killing of cells. In
E-mail address: [email protected] (V. Braun). contrast to wild-type Cma, these mutants do not prevent killing of

http://dx.doi.org/10.1016/j.ijmm.2014.12.006
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Import of Cma

Five chromosomally encoded genes are required for Cma killing

of sensitive E. coli cells, fhuA, tonB, exbB, exbD, and fkpA. Muta-
tions in any of these genes render cells resistant to high doses of
Cma (dilution titers of 105 ). The outer membrane receptor FhuA
is functionally coupled to the electrochemical gradient across the
cytoplasmic membrane via the proteins TonB, ExbB and ExbD.
These proteins are anchored in the cytoplasmic membrane and
have distinct periplasmic domains. TonB physically interacts with
FhuA, and ExbB and ExbD form a complex for which a stoichiome-
try of 6:1 was determined (Pramanik et al., 2011). It is hypothesized
that FhuA changes its conformation in response to the membrane
potential. Such conformational changes are suggested to result in
release of high-affinity ligands (Cma, ferrichrome, albomycin) from
FhuA into the periplasm. FhuA forms a ␤ barrel composed of 16 ␤
strands which is closed by an N-proximal globular segment termed
plug or cork. The plug must move to allow ligand passage through
FhuA. Currently it is unknown whether FhuA only acts as a primary
adsorption site for Cma or also as the import route. Cma uptake
is even more complex than the uptake of the other FhuA ligands
as TonB must not only interact with FhuA but also with Cma (Pilsl
et al., 1993). Cma contains a typical TonB box at the N-terminus.
Point mutations in the TonB box abolish Cma uptake. Specific point
mutations in TonB are capable to suppress point mutations in the
Cma TonB box. This is taken as an indication for a direct interaction
between the TonB box of Cma and the periplasmic domain of TonB.
Outer membrane receptor proteins and all colicins which require
TonB for activity contain an N-terminal consensus sequence, i.e. a
TonB box. Suppressor analysis, cysteine cross-linking and crystal
structures of TonB fragments bound to receptor proteins demon-
strate a physical interaction with TonB (Braun, 2014).
Fig. 1. Crystal structure (PDB 2XMX) of colicin M (Zeth et al., 2008) in which func-
tionally important residues are indicated (Helbig and Braun, 2011). N-terminal
translocation domain (yellow), central receptor binding domain (blue), C-terminal Essential role of the periplasmic chaperone FkpA for Cma
phosphatase domain (magenta),N-terminal end (N), C-terminal end (C) are indi- activity
cated. Residues identified as important for Cma activity are shown as sticks. ␣1
indicates the region involved in receptor binding. ␣2 contains a strongly polar FkpA is a peptidyl prolyl cis-trans isomerase with chaperone
sequence, 59-EDYIKKH-65, but only the alanine replacement in E59 reduces Cma
activity for which only very recently a control of outer mem-
activity to 10%.
brane biogenesis under heat shock conditions has been assigned
(Ge et al., 2014). We discovered that FkpA is essential for killing
cells by imported Cma (Hullmann et al., 2008). Likewise, FkpA is
essential for active Cma secretion into the periplasm provided an
cells by the antibiotic albomycin, a ferrichrome derivative, which artificial signal sequence has been attached to Cma (Helbig et al.,
uses FhuA to enter cells. The mutant Cma proteins do not bind to 2011). For both, import and export Cma must unfold for crossing
FhuA which indicates that the ␣1 helix mediates binding of Cma to the outer and the cytoplasmic membrane. FkpA is indispensable
FhuA. for refolding and accelerates refolding of denatured Cma in vitro.
Sequence analyses of bacterial genomes reveal cma orthologs Upon binding to FhuA Cma becomes sensitive to trypsin digestion.
in strains of Pectobacteria, Pseudomonas and Burkholderia. The A mutation analysis of the fifteen proline residues in Cma identifies
sequences are highly diverged in the receptor binding and proline P176 as the most likely one which is cis-trans isomerized.
translocation domains but similar in the phosphatase domain. Indeed, F175–P176 showed in vitro the fastest isomerization rate
The distinct receptor binding and translocation domains reflect assayed with synthetic peptides derived from the Cma sequence.
individual import proteins in these bacteria. The conserved C- P176 is exposed on the surface of the Cma crystal structure which
terminal region reflects the common killing mechanism. The was determined for wild-type Cma and the inactive P176A mutant
three-domain structure also supports our previous conclusion (Helbig et al., 2011). Of six inactive FkpA point mutants all are
that colicins in different bacteria evolved by horizontal gene located in the isomerase domain. This data identify FkpA as essen-
transfer via plasmids and an exchange of gene fragments which tial for Cma in vivo activity and suggest that Cma is unfolded during
encode functional domains (Braun et al., 2002; Roos et al., 1989). import and refolded by FkpA involving cis-trans isomerization of
The phosphatase domain evolved from a single ancestor gene the F175–P176 bond. Further, the data demonstrate an enzymatic
that was fused to distinct receptor binding and translocation activity for FkpA by which a proline bond is isomerized, and demon-
genes. strate for the first time an indispensable function of FkpA as a
Residues 226 DKYDFNASTHR236 are conserved, hydrophilic and periplasmic chaperone in protein import.
exposed at the Cma surface (Fig. 1). Amino acid replacements in this
region render Cma inactive. Particularly noteworthy are mutations Specific resistance to Cma by CbrA
D226E and D226N which completely inactivate Cma. This region is
part of the active center of Cma, and D226 most likely is directly Among Cma tolerant E. coli mutants (tolM), fkpA was identified
involved in hydrolysis (Pilsl et al., 1993; Helbig and Braun, 2011). (tolerance defined as Cma binding and, presumably, uptake, but no

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zones on plates seeded with Pst sensitive E. coli that carry the Pst
receptor of Y. pestis, termed FyuA. The crystal structure of PstRT -T4L
reveals a marked difference to Pst as the two domains are connected
by a long ␣-helix in contrast to the short unstructured linker in Pst.
Although T4L is structurally very similar to the Pst activity domain,
PstRT -T4L is not inhibited by the Pst immunity protein Pim. This
indicates that Pim is specific for the Pst activity domain. In con-
trast to the T4 lysozyme, the lysozyme variant from phage T7 when
fused to PstRT is not toxic to cells. This may be due to either failure
of uptake into the periplasm or misfolding. PstRT mediated import
shows specificity.

The activity domain of Pst must unfold to enter the

periplasm
Fig. 2. Crystal structure (4AQN) of pesticin (Patzer et al., 2012). The N-terminal
domain (NT) including the TonB box (TBB) is not visible. The translocation domain To test whether PstRT -T4L must unfold for transport cysteine
(T, red), the receptor binding domain (R, blue) and the activity domain (A, orange) residues were introduced in T4L at sites which should allow forma-
are indicated. CT: C-terminus. See text for details.
tion of disulfide bridges (Patzer et al., 2012). The PstRT -T4L chimera
was chosen because it efficiently hydrolyzes murein in vitro in
killing). An additional gene locus was found which upon overex- contrast to Pst. Three constructs with PstRT -T4L cross-linked by
pression rendered cells tolerant to Cma. Unexpectedly, the gene disulfide bridges were inactive in vivo, yet regained activity upon
responsible for tolerance was cbrA (Helbig et al., 2012) of which reduction of the disulfide bonds as they killed cells. The disulfide
very little is known. The extent of tolerance depends on the level of derivatives of Pst-T4L are active in vitro which indicates that the
CbrA expression which is controlled by the transcriptional activa- disulfide linkages do not inactivate the lysozyme function. Rather
tor CreB. Expression is particularly high in minimal media which they prevent unfolding and thus inhibit uptake.
also induce cma transcription. The basal level of CbrA synthesis To examine whether the activity domain must be specifically
already confers an 18-fold higher survival rate of Cma-treated cells translocated unfolded across the outer membrane to be able to
compared to E. coli devoid of cbrA. Upon high cbrA expression the fold into an active enzyme within the periplasm, Pst and the activ-
survival rate is increased to over 1000-fold. Since CbrA is expressed ity domain were fused to a MalE fragment that contains a signal
under similar conditions as Cma, CbrA confers an appreciable Cma sequence for secretion from the cytoplasm into the periplasm.
resistance to Cma producing and Cma sensitive cells. Sequence Expression was regulated by an arabinose promoter. Upon syn-
analysis and biochemical studies assign CbrA to FAD-dependent thesis of Mal’-Pst and Mal’-A (A, activity domain) cells lyse. This
oxidoreductases. FAD is noncovalently bound to CbrA. We found suggests that the activity domain does not require the RT domains
that CbrA renders cells resistant to osmotic shock suggesting that of Pst to fold into an active domain and that there are no specific
changes in the membrane structure may cause Cma resistance. The membrane requirements for Pst translocation and refolding.
mechanism of CbrA function remains enigmatic. The active site of Pst was deduced from the crystal structure in
comparison with T4L. Amino acid replacements at selected sites
Domain structure of pesticin (Pst) reveal that the active site residues are similar but not identical to
T4L (Patzer et al., 2012).
Most Y. pestis strains possess a small plasmid (9.5 kb) that
encodes pesticin (Pst) together with the adjacent immunity gene Secretion of the hemolysin of Serratia marcescens, a model
pim. In the producer cell the immunity protein is localized in the system for type Vb secretion
periplasm where it inactivates Pst and thus prevents killing (Pilsl
et al., 1996). Murein hydrolysis does not appear to be the only activ- S. marcescens is involved in urinary tract infections, bacteremia,
ity of Pst since cells are not immediately lysed but converted to endocarditis, keratitis, arthritis, and meningitis. One of its virulence
spheroplasts without osmoprotection. Lysis is slow but killing is factors is a hemolysin/cytolysin that lyses erythrocytes and elic-
fast. Murein hydrolysis by Pst in vitro is also slow (Vollmer et al., its inflammatory response in leukocytes (Hertle et al., 1999; König
1997). The crystal structure of Pst (Patzer et al., 2012) reveals two et al., 1987; Lin et al., 2010; Marre et al., 1989). Hemolysin was the
distinct structural domains connected by a short peptide linker first example of a by now large group of proteins which are secreted
(Fig. 2). The N-terminal domain represents the receptor binding and by the type Vb or the two-partner secretion system (TPS) (Braun
translocation domain, the C-terminal domain the activity domain. et al., 1987; Poole et al., 1988; Schiebel et al., 1989; Jaçob-Dubuisson
The structure of the activity domain is similar to phage lysozymes, et al., 2013).
in particular to that of the T4 phage, albeit sequence identity is only The hemolysin is determined by two genes, shlA and shlB,
13%. The near identity of the activity domain fold is particularly arranged in tandem; shlA encodes hemolysin, and shlB encodes
noteworthy since activity domains of the other colicins display no the outer membrane protein responsible for ShlA secretion. Both
overall structural similarity to known enzymes. proteins have N-terminal signal sequences for transport across the
cytoplasmic membrane by the Sec system.
T4 lysozyme is carried into the periplasm by the receptor ShlB not only secretes ShlA but it also activates it; in a shlB
and translocation domain of Pst deletion mutant, ShlA remains in the periplasm in an inactive
non-hemolytic form (Poole et al., 1988; Schiebel and Braun, 1989;
Since the Pst activity domain resembles T4 lysozyme (T4L) Schiebel et al., 1989). Activation can be uncoupled from secretion by
it was replaced by T4L to test whether T4L is translocated into in vitro incubation of isolated periplasmic ShlA with purified ShlB
the periplasm via Pst receptor-binding and translocation domains resulting in hemolytic ShlA (Hertle et al., 1997; Ondraczek et al.,
(PstRT ) (Patzer et al., 2012). PstRT -T4L kills cells but a 6-fold higher 1992). A ShlA N-terminal fragment of 242 residues (ShlA consists
concentration than wild-type Pst is required to yield the same lysis of 1578 residues) activates inactive periplasmic ShlA to hemolytic

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quantitative assay. The predicted structure of ShlB (Fig. 3) was used

to design deletion mutants of H1, L6, and the two POTRA domains
to study their effects on secretion and activation.
Deletion of H1 does not affect secretion of hemolytic ShlA. H1
has no essential function in ShlB assembly, secretion and activa-
tion of ShlA (Pramanik et al., 2014). Deletion of L6 diminishes,
but does not fully abolish secretion, but completely compromises
ShlA activation. The P1 and P2 single mutants secrete reduced
amounts of ShlA of which only ShlA of the P1 mutant shows some
hemolytic activity. Thus P1 and P2 mutants discriminate between
secretion and activation. H1− , P1− , P2− triple deletion mutants
secrete no ShlA. Random mutagenesis yields inactive ShlB deriva-
tives mutated in L6 which supports the importance of the conserved
L6 among type Vb transporters (Pramanik et al., 2014; Yang and
Braun, 2000).
ShlA is secreted and folds into a hemolytic structure through its
N-terminus which is secreted first. Secretion of non-hemolytic ShlA
is inefficient. Folding into the active conformation probably drives
thermodynamically secretion of ShlA through ShlB.

Acknowledgments

We thank Andrei Lupas for the generous hospitality in his

Fig. 3. Model of ShlB based on the crystal structure of FhaC (Clantin et al., 2007; department and Joachim Schultz and Klaus Hantke for critically
Pramanik et al., 2014). reading the manuscript. This work was financed by the Max Planck
Society, the German Science Foundation (BR330/25-1, SFB766) and
ShlA provided ShlA242 is secreted by ShlB (Ondraczek et al., 1992; the Fonds der Chemischen Industrie. Space limitation did not allow
Schönherr et al., 1993). ShlA242 purified from the periplasm of citing all of the relevant literature.
a shlB mutant cannot activate periplasmic ShlA. ShlA242 mimics
secretion and activation of ShlA by ShlB but is not hemolytic since
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Please cite this article in press as: Braun, V., et al., Import and export of bacterial protein toxins. Int. J. Med. Microbiol. (2015),
http://dx.doi.org/10.1016/j.ijmm.2014.12.006