Molecular Microbiology (2002) 43(4), 1005–1021
Behaviour of topological marker proteins targeted to
the Tat protein transport pathway
Nicola R. Stanley,1,2† Frank Sargent,1
Grant Buchanan,2 Jiarong Shi,3 Valley Stewart,3‡
Tracy Palmer1,2 and Ben C. Berks1,4*
1
Centre for Metalloprotein Spectroscopy and Biology,
School of Biological Sciences, University of East Anglia,
Norwich NR4 7TJ, UK.
2
Department of Molecular Microbiology, John Innes
Centre, Norwich NR4 7UH, UK.
3
Section of Microbiology, Cornell University, Ithaca, NY
14853, USA.
4
Department of Biochemistry, University of Oxford,
South Parks Road, Oxford OX1 3QU, UK.
Summary
The Escherichia coli Tat system mediates Secindependent export of protein precursors bearing
twin arginine signal peptides. Formate dehydrogenase-N is a three-subunit membrane-bound enzyme,
in which localization of the FdnG subunit to the
membrane is Tat dependent. FdnG was found in the
periplasmic fraction of a mutant lacking the membrane anchor subunit FdnI, confirming that FdnG is
located at the periplasmic face of the cytoplasmic
membrane. However, the phenotypes of gene fusions
between fdnG and the subcellular reporter genes
phoA (encoding alkaline phosphatase) or lacZ
(encoding b-galactosidase) were the opposite of
those expected for analogous fusions targeted to
the Sec translocase. PhoA fusion experiments have
previously been used to argue that the peripheral
membrane DmsAB subunits of the Tat-dependent
enzyme dimethyl sulphoxide reductase are located
at the cytoplasmic face of the inner membrane. Biochemical data are presented that instead show
DmsAB to be at the periplasmic side of the membrane. The behaviour of reporter proteins targeted to
the Tat system was analysed in more detail. These
data suggest that the Tat and Sec pathways differ in
their ability to transport heterologous passenger
proteins. They also suggest that caution should be
Accepted 9 November, 2001. *For correspondence. E-mail
[email protected]; Tel. (+44) 1865 275250; Fax (+44) 1865
275259. Present addresses: †Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles,
CA 90095-1489, USA. ‡Section of Microbiology, University of
California, One Shields Ave., Davis, CA 95616-8665, USA.
© 2002 Blackwell Science Ltd
observed when using subcellular reporter fusions
to determine the topological organization of Tatdependent membrane protein complexes.
Introduction
In Escherichia coli, the movement of most periplasmic
and membrane-bound proteins from their site of
synthesis in the cytoplasm to their final subcellular
location is mediated by the Sec protein translocation
apparatus (Pugsley, 1993). Proteins are targeted to the
Sec system by discrete, often amino-terminal regions
of polypeptide termed signal sequences. Transport
across or insertion into the membrane is by an aminoterminal to carboxy-terminal threading mechanism, in
which the substrate protein is in an extended conformation. Recently, a second general protein transport
system has been identified in the cytoplasmic membrane
of E. coli and many other bacteria (Settles et al., 1997;
Santini et al., 1998; Sargent et al., 1998; Weiner
et al., 1998). Targeting to this second pathway is by
means of signal sequences bearing a distinctive twin
arginine-containing amino acid sequence motif (Berks,
1996; Stanley et al., 2000). In contrast to the Sec
apparatus, this twin arginine (Tat) translocase functions
to transport prefolded proteins (reviewed by Berks
et al., 2000). The majority of Tat substrates in enteric
bacteria are the peripheral membrane subunits of
membrane-bound electron transfer complexes (Berks,
1996; Gennis and Stewart, 1996). Analogues of the
bacterial Sec and Tat pathways are found in the thylakoid
membrane of plant chloroplasts (Keegstra and Cline,
1999).
Gene fusions have been extensively exploited as a
tool to probe the Sec protein transport pathway and to
determine the topological organization of transmembrane
proteins assembled by the Sec apparatus (BroomeSmith et al., 1990; Manoil et al., 1990; Schatz and
Beckwith, 1990; Traxler et al., 1993; Danese and
Silhavy, 1998). This approach involves fusing a protein
fragment containing potential targeting determinants to a
reporter protein with an activity that is sensitive to subcellular location. Consequently, the activity of the reporter
protein indicates the compartment to which the protein
has been targeted. The reporter proteins typically chosen
have an easily defined phenotype in plate screens, for
example antibiotic resistance or colour development,
1006
N. R. Stanley et al.
enabling the fusion construct to be used in genetic
selection protocols. With the proven success of gene
fusion as probes of the Sec pathway, it is of interest
to explore carefully the application of gene fusions to
the genetic analysis of the Tat pathway as well as to
the topological organization of Tat-targeted enzyme
assemblages.
A consideration of the different modes of protein
translocation performed by the Tat and Sec pathways
suggests that it would be dangerous to assume that fusion strategies and reporter proteins appropriate to one
system will perform in an identical manner when applied
to the other. In at least some cases, there appears to
be a requirement that Tat substrate proteins are correctly
folded before translocation can take place (Roffey and
Theg, 1996; Santini et al., 1998; Halbig et al., 1999;
Sanders et al., 2001). If this is a general property of
the Tat pathway, then fusion proteins in which folding
is disrupted (either by removal of the carboxy-terminal
portion of the targeting domain or because addition of
the reporter protein precludes correct folding of the
targeting domain) will not be competent for Tat transport.
A similar situation appertains if the reporter protein only
folds after transport. For example, folding of the reporter
enzyme E. coli alkaline phosphatase (PhoA) requires
the formation of intramolecular disulphide bonds by a
periplasmic enzyme system (Sone et al., 1997). It is also
conceivable that the structure of the chimeric precursor
protein might impede interactions between the twin
arginine signal peptide and the transport mechanism. In
the Tat mechanism, the substrate protein is presumably
transported in a single step, implying an upper limit to the
size of the fusion proteins that can be translocated. In
contrast, the threading mechanism of the Sec pathway
probably imposes no such constraint on the substrates.
Finally, the most widely used reporter proteins are either
substrates of the Sec pathway or cytoplasmic proteins
and, thus, cannot automatically be assumed to be
competent for translocation by the Tat mechanism. For
example, the mature domains of these proteins could
recruit chaperones and interfere with Tat targeting and
transport.
Here, we show that reporter gene fusions to the
periplasmically located subunits of the Tat-dependent
and membrane-bound E. coli enzyme formate dehydrogenase-N (FDH-N) imply an incorrect topological organization of the enzyme. We then assess the behaviour of
four commonly used subcellular reporter proteins (Bla, blactamase; Cat, chloramphenicol acetyltransferase; LacZ,
b-galactosidase; and PhoA) when targeted to the Tat
pathway. We conclude that heterologous proteins cannot
necessarily be assumed to be acceptable Tat substrates
and that, in some cases, the transport behaviour is fusion
specific.
Results and discussion
The formate-oxidizing subunit of formate
dehydrogenase-N is located at the periplasmic face of
the cytoplasmic membrane
In the main portion of this study, we have sought to
determine whether standard reporter fusion constructs
can be applied to the topological mapping of membranebound enzyme complexes assembled by the Tat pathway. Our model protein in these studies was the E. coli
nitrate-inducible respiratory formate dehydrogenase
(FDH-N) encoded by the fdnGHI operon. Respiratory
formate dehydrogenases, including FDH-N, oxidize
formate to carbon dioxide concomitant with reduction
of membrane quinone to quinol. Transfer of each pair
of electrons in this reaction is linked to the net translocation of two protons from the cytoplasm to the periplasm (Kröger, 1975; Jones, 1980), and the respiratory
formate dehydrogenases therefore function as coupling
sites.
Localization of FDH-N to the cytoplasmic membrane
requires a functional Tat pathway (Bogsch et al., 1998;
Sargent et al., 1998; 1999), and the organization of the
enzyme is typical of membrane protein complexes assembled by the Tat system. In such proteins, two cofactorbinding peripheral membrane subunits complex with a
third integral membrane subunit containing the site of
interaction with the membrane quinone/quinol pool. Association of the peripheral subunit pair with the integral
membrane protein requires a Tat signal peptide located
on one of the peripheral subunits, whereas the integral
membrane subunit is thought to be inserted into the membrane by the standard SRP/Sec pathway. The peripheral
membrane subunits of FDH-N are FdnG and FdnH (Berg
et al., 1991a). Formate is oxidized at a molybdopterin
cofactor in the FdnG subunit. FdnH is a ferredoxin subunit,
and the integral membrane FdnI subunit is dihaem
cytochrome bFdn556.
In order to use E. coli respiratory formate
dehydrogenase-N (FDH-N) in our study, we needed to
be certain of the topological orientation of the enzyme.
However, the literature contains conflicting reports on the
location of the active site subunits (FdnG homologues)
in orthologous formate dehydrogenases. A periplasmic
location has previously been inferred for the formateoxidizing site of the respiratory formate dehydrogenase of
Wolinella succinogenes based on the observation that W.
succinogenes can oxidize external formate at circumneutral pH even though the cytoplasmic membrane is formate
impermeant under these conditions (Kröger et al., 1980).
In contrast, a gene fusion study of E. coli respiratory
formate dehydrogenase-O (FDH-O) assigned a cytoplasmic location to the formate-oxidizing subunit (Benoit et al.,
1998). To resolve this discrepancy, we undertook our own
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
Topological analysis of Tat substrates
biochemical analysis of the subcellular location of the
FdnG subunit.
One indicator of periplasmic localization is Tat signal
peptide cleavage. Inspection of the fdnG sequence
suggests that a 33-residue twin arginine signal peptide
is present culminating at the predicted cleavage site
Ala-Leu-Ala Ø Gln-Ala-Arg-Asn-Tyr. We determined the
amino-terminal sequence of the FdnG subunit to be
Gln-Ala-Arg-Asn-Tyr. Thus, the predicted Tat signal
peptide was post-translationally removed. The twin
arginine signal peptide is likewise cleaved from the
homologous FdhA protein of W. succinogenes formate
dehydrogenase (Bokranz et al., 1991). As the active site
of the E. coli LepB signal peptidase is located at the
periplasmic side of the cytoplasmic membrane (Dalbey
et al., 1997), the observed processing of the signal
peptide indicates that at least the amino-terminal portion
of FdnG reaches the periplasmic compartment. The
amino-terminus of the FdnH subunit was determined to
be Ala-Met-Glu-Thr-Gln, indicating that only the initiator
methionine residue was post-translationally removed,
again congruent with studies of the homologous FdhB
protein of W. succinogenes formate dehydrogenase
(Bokranz et al., 1991).
To obtain further evidence of a periplasmic localization
for FdnG, we constructed a mutant strain with a complete
in frame chromosomal deletion of the fdnI gene. Both
fdnI + and DfdnI strains were grown anaerobically to
midexponential phase on glucose plus nitrate medium to
induce FDH-N synthesis. Immunoblotting revealed intact
FdnG polypeptide in both strains. Upon subcellular fractionation, FdnG protein was found, as expected, predominantly associated with the membrane fraction of the
fdnI + strain (Fig. 1). In contrast, FdnG protein was located
almost exclusively in the periplasmic fraction of the DfdnI
Fig. 1. Deletion of the structural gene encoding the integral
membrane subunit of formate dehydrogenase-N leads to an
accumulation of the catalytic FdnG subunit in the periplasm. Cells
were cultured anaerobically on glucose- and nitrate-containing
medium to early stationary phase (OD600 = 2.5–3.0) and
fractionated. Where indicated, the membrane fraction was
additionally washed in buffer containing 0.4 M NaCl. Immunoblots of
samples separated by SDS–PAGE were probed with a polyclonal
antiserum directed against FdnG. Each lane on the gel contains
subcellular extract derived from the same wet weight of cells. p,
periplasmic fraction; c, cytoplasmic fraction; m, membranes; wm,
washed membranes.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
1007
strain (Fig. 1). The small proportion of FdnG antigen that
co-sedimented with the crude membrane fraction in the
DfdnI mutant was completely removed by a salt wash,
whereas FdnG was still present in the membranes from
the fdnI + strain after salt treatment (Fig. 1). We conclude
from this experiment that, in intact FDH-N, the FdnG
polypeptide is located on the periplasmic face of the
cytoplasmic membrane.
The formate dehydrogenase activities of the parental
and DfdnI strains were measured using phenazine methosulphonate (PMS) as electron acceptor. Whereas cells
of the fdnI + strain exhibited strong formate dehydrogenase activity, the DfdnI strain produced no detectable
formate:PMS oxidoreductase activity (data not shown).
The failure of the periplasmically located FdnG to catalyse formate:PMS oxidoreductase activity in the DfdnI
mutant could result from greater lability of the metallocofactors in the partially assembled enzyme but may, alternatively, indicate that the PMS-interacting site is located
on the FdnI and/or the FdnH subunit. In this context, it is
notable that the FDH-N protein that accumulates in the
cytoplasm of tat mutant strains is also enzymatically
inactive in the PMS-linked assay (Bogsch et al., 1998;
Sargent et al., 1998; 1999).
A model for the topological organization of FDH-N
taking account of the experimental data presented
here is given in Fig. 2A. Given that the protons derived
from formate oxidation by respiratory formate dehydrogenases are released at the periplasmic side of the
membrane and that cytoplasmic protons are used for
quinone reduction (Geisler et al., 1994), net proton
translocation by these enzymes must arise from the electrogenic movement of electrons from the periplasmic to
the cytoplasmic side of the membrane (Fig. 2A). The two
haem groups located in the integral membrane subunit
are expected to mediate the transmembrane movement
of electrons required by this scheme (Fig. 2A; Berks et al.,
1995).
The subcellular location of the FdnH protein in the
DfdnI mutant was not addressed in our experiments. Even
though FdnH lacks a cleavable signal peptide (above),
this subunit is thought to carry electrons from FdnG
to FdnI and should therefore be located at the same side
of the membrane as FdnG (Fig. 2A). This would imply
that FdnH is carried across the membrane in complex
with FdnG by a mechanism analogous to that already
described for [NiFe]-hydrogenase biosynthesis (Rodrigue
et al., 1999). This predicted complex formation suggests that FdnH should co-localize with FdnG to
the water-soluble periplasmic fraction of the DfdnI
mutant even though FdnH is probably finally secured to
the cytoplasmic membrane in the intact FDH-N complex
via a carboxy-terminal anchor (Fig. 2A; Berg et al.,
1991a).
1008
N. R. Stanley et al.
A
B
Fig. 2. Topological models for E. coli formate dehydrogenase-N and E. coli DMSO reductase.
A. Model for formate dehydrogenase-N. FdnG contains a selenocysteine-ligated molybdenum di(molybdopterin guanine dinucleotide) cofactor,
at which formate is oxidized to carbon dioxide. The electrons derived from formate are transferred by a series of iron–sulphur clusters in FdnG
and FdnH to the integral membrane subunit FdnI, where they are used to reduce menaquinone (MQ) to menaquinol (MQH2). The FdnI subunit
is a member of a sequence-related family of integral membrane proteins that bind two haem groups, one located in each half of the
membrane bilayer, and which therefore provide a possible route for transmembrane electron transport (Berks et al., 1995; Gross et al., 1998b;
Meek and Arp, 2000). Enzymes containing integral membrane proteins from the FdnI family are predicted to contain five transmembrane
helices. Four of these transmembrane helices bind the haem prosthetic groups, while the fifth transmembrane helix is distinguished only by
the presence of a conserved and functionally important (Gross et al., 1998b) histidine residue towards the cytoplasmic side of the membrane.
In many of these enzymes, including FDH-N, the fifth predicted transmembrane helix is part of one of the peripheral membrane subunits
rather than a component of the integral membrane protein. In FDH-N, the additional transmembrane helix is located at the carboxy-terminus of
FdnH.
B. Model for DMSO reductase. Two electrons released by the oxidation of MQH2 by DmsC are transferred via a series of iron–sulphur clusters
in DmsB to the molybdenum di(molybdopterin guanine dinucleotide) cofactor in DmsA, at which DMSO is reduced to dimethyl sulphide (DMS).
DmsA and DmsB are homologous to FdnG and FdnH respectively. DmsAB are shown at the periplasmic face of the inner membrane in
accordance with data presented here. In the alternative model of Sambasivarao et al., 1990), DmsAB are located at the cytoplasmic face of
the membrane. The topological organization of DmsC is based on Weiner et al. (1993).
Topological mapping of formate dehydrogenase-N using
standard genetic fusion techniques leads to an
erroneous structural model of the enzyme
To test the utility of genetic fusion technology in the topological mapping of Tat-dependent proteins, we undertook
a marker fusion analysis of formate dehydrogenase-N.
For this study, we adopted the widely used strategy of
constructing complementary fusions to the periplasmic
marker enzyme PhoA and the cytoplasmic marker
enzyme LacZ (Manoil, 1990). PhoA contains two intramolecular disulphide bonds that are essential for both
enzymatic activity and stability (Sone et al., 1997). As
the formation of disulphide bonds in wild-type E. coli cells
occurs in the periplasmic compartment, PhoA is only
active when targeted to the periplasm. LacZ is a cytoplasmic protein containing sequences that hinder transport by the Sec apparatus (Lee et al., 1989) usually
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
Topological analysis of Tat substrates
leading to a misfolded, and thus inactive, protein. The
formation of disulphide bonds within the LacZ polypeptide
as it emerges into the periplasm has been implicated in
this phenomenon (Bardwell et al., 1991). LacZ activity is
therefore normally only observed from fusions targeted
to the cytoplasm. Defined gene (translational) fusions
were constructed between each of the fdn structural
genes and phoA and lacZ separately. The resulting
plasmid-borne gene fusions are expressed under the
control of the native fdnG promoter; constructs with
fusions to fdnH include an intact upstream fdnG gene, and
those with fusions to fdnI include intact upstream fdnGH
genes. Plasmids bearing these fusion constructs were
introduced into a pcnB background to ensure a low
plasmid copy number (Lopilato et al., 1986). Reporter
enzyme activities were assessed from cultures grown
anaerobically in the presence of nitrate to induce expression from the fdnG promoter fully (Li and Stewart,
1992).
The integral membrane protein FdnI is predicted to
comprise four transmembrane helices organized such
that the amino-terminus of the protein is located at the
cytoplasmic side of the membrane (Berks et al., 1995). To
test this model, we chose to construct both phoA and lacZ
gene fusions at positions located successively in each
of the five predicted extramembranous regions (Boyd
et al., 1993). Junctions are at the distal portions of the
amino-terminal cytoplasmic tail (Asp-14), the periplasmic
loop between helices 1 and 2 (Gln-51), the cytoplasmic loop between helices 2 and 3 (Ala-111), the periplasmic loop between helices 3 and 4 (Leu-152) and the
car-boxyl-terminal cytoplasmic tail (Glu-215). The relative
activities of the resultant PhoA and LacZ fusions were
entirely consistent with the model (Table 1; Fig. 2A). Thus,
as found for many other Sec-assembled integral membrane proteins, fusions of PhoA and LacZ to FdnI gave
apparently reasonable topological information. Experiments with Bla gene fusions yielded an identical topological model for the homologous FdoI protein (Benoit et al.,
1998).
PhoA and LacZ fusions were also constructed at two
positions each in the Tat-dependent FdnG and FdnH subunits, as described in Experimental procedures. Fusions
to the FdnG polypeptide were made at codon Lys-44 in
fdnG, 11 codons downstream of the leader peptidase
cleavage site, and at codon Asp-792, about 78% into the
1016 codon gene. Fusions to the FdnH polypeptide were
made just after codons Leu-257 and His-292, which are
located on either side of the predicted transmembrane
helix.
Results from enzyme assays of cultures expressing
these fusions are shown in Table 1. All four fusion junctions yielded high-level LacZ activity and negligible PhoA
activity. In a previous study, F(fdnG–lacZ) gene fusions
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
1009
Table 1. LacZ and PhoA enzyme activities expressed from
F(fdn–lacZ) and F(fdn–phoA) gene fusions.a
Enzyme sp. act.c
Gene
Fusion junction b
fdnG
Lys-44
Asp-792
700
720
<1
<1
fdnH
Leu-257
His-292
440
870
<1
<1
fdnI
Asp-14
Gln-51
Ala-111
Leu-152
Glu-215
1720
<1
320
<1
1580
LacZ
PhoA
<1
350
<1
1800
10
a. Plasmid-bearing cultures of strain VJS5833 (Dlac DphoA pcnB1)
were grown anaerobically in TYEGN medium to mid-exponential
phase and harvested for enzyme assays.
b. fdn codon at the junction with lacZ or phoA.
c. Activities are expressed in Miller units. Values are averaged from
at least two independent experiments.
constructed at codons Ala-212 and Asp-493 also yielded
high-level LacZ activity (Berg et al., 1991b). [Conversely,
a F(fdnG–lacZ) gene fusion constructed at codon Leu32, just proximal to the leader peptidase cleavage site,
expressed very low levels of LacZ activity (Li and Stewart,
1992)]. Furthermore, in preliminary studies, an in frame
TnphoA insertion (Manoil and Beckwith, 1985) at fdnG
codon Gly-65 expressed undetectable PhoA activity (S. B.
Williams and V. Stewart, unpublished observations). The
conventional interpretation of these results would be that
both the FdnG and the FdnH polypeptides are localized
to the cytoplasm, the exact opposite of the conclusion
drawn from the studies of leader peptide cleavage and
periplasmic FdnG accumulation described above. Indeed,
analysis of Bla fusions to the homologous FdoG and
FdoH proteins of E. coli FDH-O were interpreted to reveal
a cytoplasmic location for these subunits (Benoit et al.,
1998). In summary, analysis of three conventional topology reporters (Bla, LacZ and PhoA) resulted in predictions
that are directly contradicted by the results of the biochemical studies presented above.
Investigating the behaviour of Tat-targeted
reporter proteins
The unexpected pattern of whole-cell activities measured
for FdnG–PhoA and –LacZ fusions implies that either the
addition of a Tat signal peptide to these reporter enzymes
is insufficient to allow export by the Tat pathway or the
enzymes themselves are incompatible with Tat transport.
In order to investigate this phenomenon in more detail, we
undertook a biochemical analysis of the behaviour of different passenger proteins fused to the FdnG twin arginine
signal peptide. As well as PhoA and LacZ, we selected
1010
N. R. Stanley et al.
Table 2. Enzymatic activities of fdnG translational fusions after subcellular fractionation.
Activities of fdnG fusion constructs
Strain
Subcellular fraction
F(fdnG–lacZ )K44
F(fdnG–bla)K44
F(fdnG–bla)D792
F(fdnG–cat)K44
MC4100
Periplasm
Cytoplasm
Membranes
0.5 ± 0.1 (1%)
54 ± 5 (98%)
0.7 ± 0.1 (1%)
1.6 ± 0.1 (80%)
0.36 ± 0.01 (18%)
0.04 ± 0.01 (2%)
1.6 ± 0.1 (80%)
0.4 ± 0.1 (20%)a
0.6 ± 0.1 (69%)
0.20 ± 0.01 (23%)
0.07 ± 0.01 (8%)
JARV16
(DtatADtatE)
Periplasm
Cytoplasm
Membranes
0.4 ± 0.1 (2%)
18 ± 3 (84%)
3.0 ± 0.3 (14%)
ND
ND
ND
1.5 ± 0.1 (75%)
0.5 ± 0.1 (25%)a
0.09 ± 0.01 (7%)
1.2 ± 0.2 (88%)
0.07 ± 0.01 (5%)
Cells were cultured anaerobically on glucose- and nitrate-containing medium to early stationary phase (OD600 = 2.5–3.0) and fractionated as
described in Experimental procedures. Activities are given as mmol of substrate hydrolysed min–1 for the volume of the subcellular fraction obtained
from 1 g wet weight of cells. The substrates used in these assays are listed in Experimental procedures. All assays were performed on three
independent cultures with errors representing SEM. The figures in brackets give the percentage of the total enzymatic activity associated with
each subcellular compartment.
ND indicates that the experiment could not be performed (see text).
a. The activity is that of lysed spheroplasts (i.e. cytoplasmic and membrane fractions combined).
Bla and Cat as passenger proteins for use in these
studies. The reporter proteins Bla and Cat provide genetic
selections for fusion proteins that are, respectively,
exported to the periplasm (the site of b-lactam action) or
retained in the cytoplasm (where chloramphenicol is inactivated by acetylation) (Zelazny and Bibi, 1996; reviewed
by Broome-Smith et al., 1990). All four types of fusion
were constructed at fdnG codon Lys-44, resulting in
hybrid proteins containing the FdnG signal peptide
together with the first 11 residues of the mature protein.
In the experiments described in this, and indeed all other,
sections of this report, the quality of the subcellular fractionation was confirmed by an analysis of marker enzyme
distribution as detailed in the Experimental procedures.
Alkaline phosphatase activity was undetectable in cells
harbouring the F(fdnG–phoA)K44 fusion plasmid (Table 1).
In addition, we were unable to detect PhoA-immunoreactive material in these cells (data not shown), possibly
because cytoplasmically targeted PhoA is unfolded and
prone to proteolytic degradation. PhoA protein and alkaline phosphatase activity were also undetectable in tat
mutant strains containing the F(fdnG–phoA)K44 construct.
This observation argues against the possibility that the
fusion is transported by the Tat system and subsequently
proteolysed in the periplasm as, if this were the case, one
might expect the fusion protein to accumulate in the
cytoplasmic fraction of the tat mutants.
The F(fdnG–lacZ )K44 construct expressed active LacZ
enzyme (Table 1). Upon subcellular fractionation, both
LacZ activity (Table 2) and LacZ-immunoreactive
material (data not shown) were found exclusively in the
cytoplasmic fraction. Thus, the LacZ fusion was either
not exported or was unstable in the periplasm after export.
Targeting of LacZ to the Sec pathway can result in
jamming of the Sec translocase (Ito and Beckwith, 1981),
and it was therefore conceivable that an analogous
phenomenon was preventing export of the Tat-directed
LacZ fusion in our experiments. Expression of the
F(fdnG–lacZ)K44 fusion had no effect on the levels of
FDH-N assembled in the membrane or on targeting of the
Tat substrate trimethylamine N-oxide reductase (TorA)
to the periplasm (data not shown), suggesting that, under
these experimental conditions, the fusion did not block the
Tat pathway. However, expression of the F(fdnG–lacZ)K44
fusion in a pcnB + background (in which the encoding
plasmid is present at high copy number) led to a substantial (80%) drop in periplasmic TorA levels. Taken
together, these observations suggest that, although
the hybrid protein did not jam the Tat translocase, it
did compete with endogenous substrates for access to
the Tat transport system. A corollary is that the Tat
system must recognize the Tat signal peptide of the
F(fdnG–lacZ)K44 fusion and, therefore, that the failure to
transport LacZ is unlikely to be a consequence of proteolysis or other occlusion of the Tat signal peptide on the
fusion.
Fractionation of cells expressing the F(fdnG–bla)K44
fusion revealed that around 80% of the total Bla activity
accumulated in the periplasmic fraction (Table 2).
Immunoblotting confirmed that the Bla protein was
present in the periplasmic fraction (Fig. 3). The FdnG Tat
signal peptide is therefore capable of directing Bla export.
It proved difficult to test whether this export was mediated
by the Tat system, as tat mutant strains lysed when
expression of the F(fdnG–bla)K44 fusion was induced in
liquid culture. However, the F(fdnG–bla)K44 fusion conferred ampicillin resistance (100 mg ml–1) to a Tat+ strain,
but not to a DtatC mutant, when plated on solid
medium. These experiments indicate that the F(fdnG–
bla)K44 fusion was transported to the periplasm via the Tat
pathway. The analogous fusion to the FDH-O orthologue
[F(fdoG–bla)R36; Benoit et al., 1998] conferred a moderate level of resistance to ampicillin [minimal inhibitory concentration (MIC) = 25 mg ml–1], which was higher than that
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
Topological analysis of Tat substrates
Fig. 3. Detection of TEM b-lactamase protein in subcellular
fractions of cells expressing fdnG–bla translational fusions. Cells
were cultured anaerobically on glucose- and nitrate-containing
medium to early stationary phase (OD600 = 2.5–3.0) and
fractionated into periplasmic and spheroplast fractions by osmotic
shock. Immunoblots of samples separated by SDS–PAGE were
probed with a polyclonal antiserum directed against b-lactamase.
The quantity of periplasmic and spheroplast fractions loaded in
each lane is prepared from the same wet weight of cells. Lane 1,
MC4100; lane 2, MC4100 pVJS2248(F(fdnG–bla)K44); lane 3,
MC4100 pNR12(F(fdnG–bla)D792); lane 4 MC4100 pBluescript-II
KS (bla).
for any other F(fdoG–bla) fusion tested but about 10-fold
lower than that for active F(fdoI–bla) fusions.
Upon subcellular fractionation of cells expressing a
F(fdnG–cat)K44 fusion, the bulk of both enzymatic activity
and Cat protein accumulated in the periplasmic fraction
(Table 2 and Fig. 4). Thus, Cat, like Bla, was targeted
for export by the FdnG signal peptide. Interestingly, although the spheroplasts retained substantive Cat activity,
this cytoplasmically located Cat was undetectable by
immunoblotting. This suggests that the exported Cat has
a substantially lower specific activity than the material that
is retained in the cytoplasm. Transport of the fusion was
abolished in a DtatADtatE background (Table 2), demonstrating that Cat was being transported by the Tat
pathway.
Taken together, these data suggest that, although the
FdnG signal peptide was able to target the heterologous
Bla and Cat proteins to the Tat transporter, some other
passenger proteins, including LacZ and PhoA, may be
inherently incompatible with the Tat system.
Behaviour of marker proteins targeted to the Tat system
by the signal peptide of the E. coli SufI protein
To test whether the observed transport behaviour of the
FdnG signal peptide fusions was signal peptide specific
or represented a more general feature of the interaction
of the passenger proteins with the Tat system, we individually fused PhoA, LacZ and Bla directly behind the
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
1011
27-amino-acid signal peptide of the E. coli SufI protein.
SufI is a water-soluble and, therefore, unambiguously
periplasmic Tat substrate (Stanley et al., 2000).
The F(sufI–phoA)A27 fusion was enzymatically active,
and the PhoA protein was detected almost exclusively
in the periplasmic fraction (data not shown). However,
the fusion was still active in a DtatC background,
indicating that the observed export did not require
the Tat system. We note that this Tat-independent
transport occurs even though the SufI signal peptide contains a c-region basic residue, a feature that has been
shown to inhibit the interaction of other Tat signal peptides
with the Sec apparatus (Bogsch et al., 1997; Cristóbal
et al., 1999).
Subcellular fractionation of E. coli cells expressing
the F(sufI–lacZ )A27 fusion showed that >90% of the
LacZ activity localizes to the cytoplasmic fraction in either
a Tat+ or a Tat– background (Table 3). Thus, within
the experimental error of this study, the SufI signal
peptide does not direct Tat-dependent export of LacZ.
The low level of expression of this fusion precluded
the detection of LacZ-immunoreactive material by
immunoblotting.
Escherichia coli strain MC4100 transformed with the
F(sufI–bla)A27 fusion plasmid was resistant to 125 mg ml–1
ampicillin on plates, whereas a DtatC derivative of
MC4100 containing the same plasmid was ampicillin sensitive. Thus, the SufI signal peptide, like the FdnG signal
peptide, is capable of directing Tat-specific export of Bla.
The low level of expression of the F(sufI–bla)A27 fusion
from the sufI promoter precluded accurate analysis of
Fig. 4. Detection of chloramphenicol acetyltransferase protein in
subcellular fractions of cells expressing fdnG–cat translational
fusions. Cells were cultured anaerobically on glucose- and nitratecontaining medium to early stationary phase (OD600 = 2.5–3.0) and
fractionated into periplasmic and spheroplast fractions by osmotic
shock. Immunoblots of samples separated by SDS–PAGE were
probed with a polyclonal antiserum directed against
chloramphenicol acetyltransferase. The quantity of periplasmic and
spheroplast fractions loaded in each lane is prepared from the
same wet weight of cells. Lane 1, MC4100
pVJS2245(F(fdnG–cat)K44); lane 2, MC4100
pNR16(F(fdnG–cat)D792); lane 3, MC4100; lane 4, MC4100
pMAK705(cat).
1012
N. R. Stanley et al.
Table 3. Enzymatic activities of sufI–lacZ translational fusions after
subcellular fractionation.
Subcellular
fraction
Activity of F(sufI–lacZ )A27 fusion
(nmol ONPG hydrolysed min–1 g–1)
MC4100
Periplasm
Cytoplasm
Membranes
5
50
£1
JARV16
(DtatADtatE)
Periplasm
Cytoplasm
Membranes
£1
8
£1
Strain
Cells were cultured aerobically on LB-glucose medium to early
stationary phase and fractionated as described in Experimental
procedures. Activities are given as nmol of substrate hydrolysed
min–1 for the volume of the subcellular fraction obtained from 1 g wet
weight of cells.
the subcellular localization of Bla by enzyme assay or
immunoblotting.
In summary, the SufI signal peptide fusions provide examples of the same signal peptide directing Tatdependent export, Tat-independent export or no export,
depending on the passenger protein. Hybrids in which
PhoA was fused to the signal peptides of FdnG and SufI
showed different transport behaviour resulting, at least in
part, from differences in the ability of these Tat signal
peptides to interact with the Sec system.
Behaviour of reporter fusions within the mature
sequence of a Tat substrate
The experiments described above show that hybrid proteins in which Cat or Bla are fused close behind the Tat
signal peptide of FdnG are capable of directing translocation of the passenger protein to the periplasm. We
decided to investigate whether there was a difference in
transport behaviour if the reporter proteins were fused
further into the FdnG protein leaving a region of misfolded
protein between the Tat signal peptide and the reporter
protein. We chose to make Cat and Bla fusions after
FdnG codon 792. This construct removes ª 22% of
the FdnG polypeptide and would destroy the fourth
consensus domain found in homologous molybdopterin
cofactor-binding structures (Boyington et al., 1997).
Strains expressing the fusions would also lack stoichiometric amounts of the partner FdnH subunit. However,
cofactor binding to the truncated FdnG protein might
still be feasible as, by analogy with related molybdopterinbinding structures, the deleted portion of FdnG
provides few contacts with the molybdopterin cofactor,
whereas all the iron–sulphur cluster-binding domain I is
still present.
Expression of the F(fdnG–bla)D792 construct resulted in
an accumulation of b-lactamase activity in the periplasmic
fraction (Table 2). However, the periplasmic localization of
the b-lactamase activity was unaffected in a DtatADtatE
mutant, indicating that export by a non-Tat pathway was
occurring (Table 2). Immunoblotting showed that the
periplasmically located Bla molecules have a molecular
mass similar to that of the native mature protein, whereas
the cytoplasmically located Bla fusion precursor protein is
subject to proteolytic degradation (Fig. 3). It is not clear,
therefore, whether the species that is transported is the
full-length fusion protein or a truncated fragment.
No chloramphenicol acetyltransferase activity could be
detected in extracts of cells expressing the F(fdnG–
cat)D792 fusion. However, the hybrid protein was detected
in the cytoplasmic fraction by immunoblotting. This protein was present primarily as a high-molecular-mass
species that showed some degradation to a form with
an electrophoretic motility close to that of wild-type Cat
(Fig. 4). As Cat activity relies on the formation of a
homotrimeric structure (Leslie et al., 1988), the lack of
activity exhibited by the fusion protein may indicate that
the Cat domains of the hybrid protein are unable to
oligomerize.
In summary, these experiments demonstrate that a
reporter protein that is targeted to the Tat pathway when
fused to a Tat signal peptide alone is not necessarily
transported when the fusion junction is within the mature
portion of a multisubunit metalloprotein using the same
signal peptide.
The DmsAB subunits of E. coli DMSO reductase
are located at the periplasmic face of the
cytoplasmic membrane
Escherichia coli enzyme dimethyl sulphoxide (DMSO)
reductase is a three-subunit membrane-bound enzyme in
which the two peripheral membrane subunits, DmsAB,
are homologous to the FdnGH subunits of FDH-N, and
the DmsC subunit is an integral membrane protein
(Weiner et al., 1992). The site of DMSO reduction is a
molybdopterin cofactor in the DmsA protein. Targeting of
the DmsAB subunits into the mature enzyme complex has
been shown to require a functional Tat system as well
as the processing of the Tat signal peptide found on the
DmsA subunit (Weiner et al., 1998; Sambasivarao et al.,
2000). Notwithstanding the involvement of the Tat system
in the biosynthesis of DMSO reductase, it has been
suggested that the DmsAB subunits are located at the
cytoplasmic rather than the periplasmic face of the membrane and that DMSO reductase is the archetype of a
subclass of Tat substrates for which the Tat apparatus
is involved solely in membrane targeting and not in
protein translocation (Weiner et al., 1998; Sambasivarao
et al., 2000; 2001). The inferred cytoplasmic localization
of DmsAB is based in part on the results of certain
types of biochemical analysis, but also on the failure to
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
Topological analysis of Tat substrates
1013
Table 4. DMSO reductase activity localizes to the periplasm in a DdmsC mutant.
Fraction
LCB628 (tor)
mmol BV ·+ oxidized min–1 (g cells)–1
TP051 (tor DdmsC)
mmol BV ·+ oxidized min–1 (g cells)–1
GB2301 (tor DdmsABC)
mmol BV·+ oxidized min–1 (g cells)–1
Periplasm
Membrane
Cytoplasm
0.9
8.4
0.7
8.1
1.3
2.3
1.0
1.9
1.1
Cells were cultured in CR medium with 0.5% glycerol, 0.5% fumarate, 0.4% DMSO. Activities were measured with TMAO as the electronaccepting substrate.
observe active phoA fusions to either dmsA or dmsB
(Sambasivarao et al., 1990), an approach that has been
shown here to be flawed. The subcellular localization of
DmsA and DmsB has also been probed by genetically
removing the integral membrane DmsC subunit. An initial
report using this strategy found that DmsA and DmsB
were located in the periplasm (Weiner et al., 1998), suggesting that DmsAB are normally found at the periplasmic side of the membrane (Sargent et al., 1998).
However, in subsequent work using the same approach,
only cytoplasmic DmsA and DmsB were detected
(Sambasivarao et al., 2001).
Establishing the true topological organization of DmsAB
is of fundamental importance in understanding the
physiological role and mechanism of the Tat pathway as,
if DmsAB are indeed located at the cytoplasmic face of
the membrane, then there is a subset of Tat substrates
for which the pathway has only a membrane targeting, as
opposed to a transport, function (Weiner et al., 1998).
We have therefore undertaken an independent assessment of the effect of expressing DmsAB in the absence
of DmsC.
DMSO reductase is encoded by the dmsABC operon.
A strain, TP051, was constructed containing a complete
in frame deletion of dmsC together with the addition of a
hexahistidine coding sequence to the 3¢ end of dmsB.
Both TP051 and the parental strain LCB628 were cultured
anaerobically in the presence of DMSO to induce expression from the dmsA promoter. The cells were fractionated,
and DMSO reductase activity was measured using the
non-physiological electron donor benzyl viologen radical
(BV•+). DMSO reductase activity in the parental strain was,
as expected, mainly (84%) present in the membrane fraction (Table 4). In contrast, the BV•+-dependent DMSO
reductase activity was found predominantly (69%) in
the periplasm of the DdmsC strain (Table 4). A control
fractionation of a DdmsABC strain suggested that the
residual DMSO reductase activity found in the cytoplasmic and membrane fractions of the DdmsC mutant does
not arise from DmsAB (Table 4). As the molybdopterin
cofactor in DmsA is the site of DMSO reduction, these
activity data indicate that the DmsA subunit is localized to
the periplasm in the absence of the DmsC membrane
anchor protein.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
Fig. 5. DmsB is located in the periplasm in a strain lacking the
DmsC membrane anchor protein. Subcellular fractions from dmsBhis
DdmsC strain TP051 and the parental strain LCB628, both
harbouring plasmid pACYC184 (cat), were subjected to
SDS–PAGE and then immunoblotting. Periplasmic (P), total
membrane (M) and cytoplasmic (C) fractions were analysed as
indicated.
Top. The blot was probed with an antiserum directed against the
hexahistidine tag that had been engineered onto the DmsB subunit
in strain TP051. The arrow indicates the position of the histidinetagged DmsB protein.
Bottom. A control for the quality of the subcellular fractionation that
has been probed with an antiserum directed against the plasmidencoded cytoplasmic protein Cat. The arrow indicates the position
of the Cat protein.
The subcellular localization of the DmsB subunit in the
DdmsC mutant was also probed using antibodies directed
against the hexahistidine tag that we had engineered onto
DmsB in the mutant. The tagged DmsB protein was found
predominantly in the periplasmic fraction (Fig. 5). This
observation indirectly supports a periplasmic location for
DmsA, as DmsB does not have a signal peptide and
would need to be transported in complex with DmsA
(Berks, 1996).
Taken together, these experiments show that both
DmsA and DmsB are directed to the periplasm of a
DdmsC mutant. The most reasonable interpretation of
these data is that DmsAB are located at the periplasmic
face of the membrane in the native DMSO reductase
complex.
Peripheral membrane subunits of other membranebound Tat complexes have been found to be released into
the periplasm upon removal of the membrane anchor
protein (FdnG, this work; Krafft et al., 1995; Bernhard
et al., 1997; Gross et al., 1998a). In these studies, as in
1014
N. R. Stanley et al.
our experiment, the membrane subunit was eliminated
by an in frame deletion of the encoding chromosomal
gene. This approach ensures that expression of the
peripheral membrane proteins is maintained at physiological levels. In contrast, in the earlier study of DmsAB
localization (Sambasivarao et al., 2001), dmsAB were
expressed from a multicopy plasmid in a complete dms
deletion background. It is possible that the Tat system
cannot cope with such high levels of DmsAB expression
and that this led to the observed cytoplasmic accumulation of DmsAB.
To test the subcellular location of DmsAB further, we
took advantage of the observation that DMSO reductase
will use trimethylamine N-oxide (TMAO) as an alternative
substrate to DMSO both in vitro and in vivo (Weiner et al.,
1988; 1992). In E. coli and other bacteria, TMAO is predominantly reduced by periplasmic water-soluble enzyme
systems. However, in an E. coli strain devoid of periplasmic TMAO reductase (TorA), the membrane-bound
DMSO reductase will still support anaerobic growth with
TMAO as the sole respiratory electron acceptor (Weiner
et al., 1992). TMAO falls into a class of organic compounds for which no completely uncharged Lewis structure can be written. Thus, TMAO is a relatively polar
molecule, and we reasoned that the cytoplasmic membrane bilayer might have a very limited permeability to this
species. With this in mind, we devised an experiment
to test the cellular location of Dms-dependent TMAO
reduction in whole cells using the membrane-permeant
molecule BV·+ (Jones and Garland, 1977) as the electron
donor. To avoid interference from the periplasmic TMAO
reductase (TorA), the experiments were carried out in a
tor background.
BV·+-dependent TMAO reductase activity can be measured in intact cells of a tor strain, and this activity is
essentially unchanged after cell disruption (Table 5). This
TMAO reductase activity is abolished in a tor dms double
mutant, indicating that the activity can be attributed to
DMSO reductase (Table 5).
All workers are agreed that DmsA and DmsB accumulate in the soluble cytoplasmic fraction of tatB
mutants (Chanal et al., 1998; Weiner et al., 1998; Sargent
et al., 1999; Sambasivarao et al., 2001). Negligible BV·+dependent TMAO reductase activity was observed in
whole cells of a tor tatB double mutant (Table 5). However,
upon cell lysis, TMAO reductase activity similar to that of
the Tat+ Tor– strain is measured (Table 5). This clearly
shows that TMAO is normally membrane impermeant, as
the substrate can only access the unequivocally cytoplasmically located DmsAB when the membrane barrier
is broken.
Our observation that the inner membrane provides a
permeability barrier to TMAO is confirmed by experiments
with strains synthesizing TorA but not Dms. BV·+-linked
Table 5. Assessing the subcellular location of DMSO reductase
using a membrane-impermeant enzyme substrate.
Enzymatic activity mmol BV·+
oxidized min–1 (g cells)–1
Strain (relevant genotype)
Intact cells
LCB628
(tor –, dms +, tat +)
GB2301
(tor –, dms –, tat +)
GB2303
(tor –, dms +, tatB)
DSS401
(tor +, dms –, tat +)
GBKK22
(torA [R11K,R12K], dms –, tat +)
12
0.5
<0.1
80
5.1
Crude extract
11
3.1
11
166
147
Cells were grown in CR medium with 0.5% glycerol, 0.5% fumarate,
0.4% TMAO, and activities were measured with TMAO as substrate.
Cells were broken by sonication in a nitrogen-saturated buffer
comprising 50 mM Tris-HCl, pH 7.5, 2.5 mM Na2EDTA, 5 mM 2mercaptoethanol and Roche protease inhibitor cocktail.
TMAO reductase activity is easily measured for intact
cells of a strain in which the water-soluble TorA is targeted
to its normal periplasmic location (Table 5). In contrast,
if the twin arginine motif of the signal peptide of the
TorA precursor is mutated such that enzymatically active
TorA is retained in the cytoplasm (strain GBKK22 in which
the conserved arginine pair of the signal peptide is substituted by two lysine residues; Buchanan et al., 2001),
then substantive BVÑ+-dependent TMAO reductase activity is only measured for broken rather than intact cells
(Table 5).
The demonstration that TMAO is poorly membrane permeant, together with the observation that the full BVÑ+dependent TMAO reductase activity of DMSO reductase
can be measured in intact Dms+ Tor– cells, indicates
that Dms-dependent TMAO reduction is a periplasmic
process. The new biochemical evidence presented here,
together with spin-coupling studies (Weiner et al., 1993;
Rothery and Weiner, 1996) and the invalidation of the
PhoA fusion approach (above), overwhelmingly point to a
location of the DmsAB subunits of E. coli DMSO reductase at the periplasmic face of the inner membrane. As
a consequence, the previous view that the Tat system
can function in membrane targeting alone is no longer
sustainable. A model for the topological organization of
DMSO reductase taking account of the data presented
here is shown in Fig. 2B.
Concluding remarks
We have examined the behaviour of gene fusions
between the signal peptides of two Tat-dependent proteins, respiratory formate dehydrogenase-N and SufI
protein, and four different passenger proteins. We found
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
Topological analysis of Tat substrates
that a Tat signal peptide can direct the Tat-dependent
export of Bla or Cat but not of LacZ or PhoA. A consideration of the three-dimensional structures of the four
marker proteins (Leslie et al., 1988; Kim and Wyckoff,
1991; Jelsch et al., 1993; Jacobson et al., 1994) indicates
that the signal peptide should be accessible at the surface
of the folded molecule even if, as expected for Cat and
LacZ, the protein has oligomerized. This suggests that the
failure to transport LacZ or PhoA through the Tat pathway
resulted from an innate incompatibility between these proteins and the Tat system. In the case of LacZ, it is probable that the fully assembled LacZ tetramer is too large
to be transported by the Tat machinery, as this molecule
is bigger (minimal cross-sectional dimensions of ª 90 Å
by 130 Å) than known substrates of the E. coli Tat system
(diameters of about 60–70 Å; Berks et al., 2000). The
inability of the Tat system to translocate LacZ is likely to
be a general phenomenon, as this protein was also not
exported in Zymomonas mobilis when fused to the signal
peptide of the Tat substrate glucose-fructose oxidoreductase (Halbig et al., 1999). As observed here for the FdnG
and SufI chimeras, both active (Keon and Voordouw,
1996) and inactive (Sambasivarao et al., 1990; Reinartz
et al., 1998) fusions have been reported between PhoA
and Tat substrates. As the activity of the F(sufI–phoA)A27
fusion was shown to be Tat independent, these experiments suggest that PhoA is incompatible with the Tat
pathway in most, and possibly all, fusion contexts. This
incompatibility may be related to the inability of PhoA to
fold in the cytoplasm, unlike Bla (Plückthun and Knowles,
1987) or the native cytoplasmic proteins Cat and LacZ.
The observation that Cat can be translocated by the Tat
pathway is intriguing, as there are reports that this protein
is not transported when targeted to the Sec system in
E. coli or Bacillus subtilis (Gentz et al., 1988; Chen and
Nagarajan, 1993). Cat may therefore be the first example
of a cytoplasmic protein that is competent for transport
through the Tat but not the Sec pathway. We note,
however, that green fluorescent protein is folded and
active when targeted to the periplasm via the Tat pathway,
but is misfolded and inactive when it has been translocated by the Sec system (Feilmeier et al., 2000; Santini
et al., 2001; Thomas et al., 2001).
One goal of the research reported here was to investigate the utility of reporter enzymes in the genetic analysis of the Tat pathway. In principle, the F(fdnG–cat)K44
fusion could be used to select for tat mutants, as the
mutant strains would become chloramphenicol resistant.
However, under the experimental conditions used here,
some active Cat fusion protein is retained within the cytoplasm at steady state (Table 2), and this is sufficient to
render wild-type cells chloramphenicol resistant (data not
shown). Further development of the system would therefore be necessary before Cat fusions could be applied
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
1015
to the genetic analysis of the Tat pathway. Bla was also
found to be specifically transported by the Tat pathway.
As a consequence, the ampicillin resistance conveyed by
the F(fdnG–bla)K44 and F(sufI–bla)A27 fusions described
here could provide a positive selection for suppressors
of tat mutations. There are, however, potential problems
with the general use of Bla as a reporter for Tat activity,
because transport of the fusions is not always totally
Tat dependent. For example, the F(fdnG–bla)D792 fusion
exhibited Tat-independent translocation, whereas the
Tat signal peptide–Bla hybrid described by Nivière et al.
(1992) was still transported when one of the signal
peptide consensus arginine residues was mutated to an
acidic residue, even though only lysine substitutions have
ever been observed to allow export of native Tat substrates (Dreusch et al., 1997; Gross et al., 1999; Halbig
et al., 1999; Stanley et al., 2000; Hinsley et al., 2001). An
additional difficulty for certain types of experiment is that
expression of the F(fdnG–bla)K44 fusion in a tat background liquid culture led to cell lysis.
Experimental procedures
Bacterial strains, plasmids and growth conditions
The strains and plasmids used in this work are summarized
in Table 6. During all genetic manipulations, and during the
biochemical analysis of sufI fusions, E. coli strains were
grown aerobically in Luria–Bertani medium (Sambrook et al.,
1989), and antibiotics were added at the concentrations listed
by Sargent et al. (1998). In order to ensure synthesis of proteins from the fdnG promoter, biochemical studies were
carried out on cells cultured anaerobically in the modified
Cohen and Rickenberg medium described by Sargent et al.
(1998) supplemented with 0.4% (w/v) glucose and 0.4% (w/v)
KNO3. Cultures for the experiment presented in Table 1 were
grown anaerobically in TYEGN medium, which consists of
Vogel–Bonner defined medium (E salts; Maloy et al., 1996)
supplemented with 0.8% tryptone, 0.5% yeast extract, 10 mM
glucose and 40 mM NaNO3.
Genetic constructs
Strain NRS-7 was constructed as follows. A 654 bp polymerase chain reaction (PCR) product covering the region
directly upstream of fdnI and the first 8 bp of the fdnI coding
region was amplified from MC4100 DNA using the primers
5¢-GCGCtctagaGGTTTACGATAACCCCGCCG-3¢ (restriction
site in lower case) and 5¢-GCGCggatccTTACTCATGAT
GATCCTCCTCG-3¢ (start codon underlined). The resulting
product was digested with XbaI and BamHI and cloned into
pBluescript-KS II to generate plasmid pNR38. The production of an equivalent PCR fragment covering the
region directly downstream of fdnI proved problematic, as this
non-coding region contains a region of repetitive DNA that
prevented faithful replication of this region. Instead, we used
a 563 bp fragment starting a full 194 bp from the fdnI
stop codon and amplified using the primers 5¢-GCGCatc
1016
N. R. Stanley et al.
Table 6. Strains and plasmids used in this study.
Strain
Genotype or description
Reference
–
Casadaban and Cohen (1979)
Sargent et al. (1999)
Liu and Parkinson (1989)
Wilmes-Riesenberg and Wanner (1992)
P1 transduction (BW13745 ¥ RP7947)
This study
Méjean et al. (1994)
This study
GB2301
MCMTA
GB2303
F DlacU169 ara139 rpsL150 relA1 ptsF rbs flbB5301
MC4100 DtatA DtatE
pcnB1 zad-981::Tn10d(Km) leuB6 fhuA
D(lacI-codB)X74 DphoA532
BW13745 pcnB1 zad-981::Tn10d(Kmr)
MC4100 DfdnI
MC4100 torC::W Specr
LCB628 DdmsC and dmsB with hexahistidine tag
coding region at 3¢ end
MC4100 Ddms::Kanr
DSS401 with torA allele bearing Lys for Arg codon
substitutions at codons 11 and 12
LCB628 Ddms::Kanr
MC4100 tatB::Kanr
LCB628 tatB:: Kanr
Plasmids
pVJS2213
pVJS2248
pVJS2245
pVJS2216
pNR12
pNR16
pMAK705
pBluescript-KS II
pACYC184
pQE60
pDHB5700
pSK4158
pHH18
pHH21
pHH26
F(fdnG-lacZ)K44
F(fdnG–bla)K44
F(fdnG–cat)K44
F(fdnG–phoA)K44
F(fdnG–bla)D792
F(fdnG–cat)D792
Cmr, lac¢ IOPR¢
General cloning vector, Ampr
General cloning vector, Chlr
Cloning vector for addition of C-terminal hexahistidine tag. Ampr
Kanr
Cmr, ¢phoA
F(sufI–bla)A27
F(sufI–phoA)A27
F(sufI–lacZ)A27
This study
This study
This study
This study
This study
This study
Hamilton et al. (1989)
Stratagene
Rose (1988)
Qiagen
G. von Heijne
Paulsen et al. (1995)
This study
This study
This study
MC4100
JARV16
RP7947
BW13745
VJS5833
NRS-7
LCB628
TP051
DSS401
GBKK22
gatATAACAGTTTCAAATGGCGCTGT-3¢ (which introduces a
stop codon, underlined, in frame with fdnI in resulting plasmid
pNR70) and 5¢-GCGCggtaccGCGTAGGAAGGAACAATAA
TG-3¢. This was digested with ClaI and KpnI and ligated into
pNR38 to produce plasmid pNR70. The complete insert was
then removed by XbaI and KpnI digestion and ligated into
pMAK705 for integration into the chromosome according to
the method of Hamilton et al. (1989).
Strain TP051 was constructed as follows. A 668 bp fragment covering the region directly preceding the stop codon
of dmsB was amplified from MC4100 DNA using primers
5¢-GCGCgaattcCGTCACATACAAACCTTGTTCAGG-3¢ and
5¢-GCGCagatctCACCTCCTTCGGGTTTGCCAG-3¢,
digested with EcoRI and BglII and cloned into pQE60 to
generate plasmid pDMSB1. DNA covering the stop codon
of dmsC and the downstream 544 bp was amplified from
MC4100 DNA using primers 5¢-GCGCaagcttTAATCATAA
CAACCGGGGTTTCGG-3¢ and 5¢-GCGCatcgatCGTGCTG
GCGCTGGGTGACCTGGC-3¢, digested with HindIII and
ClaI and cloned into pBluescript-KS II to give plasmid
pDMSDEL1. The DNA covering the his-tagged DmsB region
was excised from plasmid pDMSB1 by digestion with EcoRI
and HindIII and cloned into pDMSDEL1 to give plasmid
pDMSDEL2. The DNA covering the his-tagged DmsB-deleted
DmsC region was excised from pDMSDEL2 by digestion with
KpnI and XbaI and cloned into the polylinker of pMAK705
to give plasmid pDMSDEL3. The mutant allele was recombined into the chromosome of LCB628 using the method of
Hamilton et al. (1989).
Sambasivarao and Weiner (1991)
Buchanan et al. (2001)
P1 transduction (LCB628 ¥ DSS401)
Chanal et al. (1998)
P1 transduction (LCB628 ¥ MCMTA)
Gene (translational) fusions to the fdnG gene were made
at a BamHI site that was engineered by site-specific mutagenesis at codon Lys-44 in fdnG (44-AAAGAGATC-46
changed to 44-AAGGATCCC-46), and at a native BamHI
site overlapping codon Asp-792 (791-TGGGATCCG-793).
Fusions to the fdnH gene were made at an engineered SalI
site at codon Lys-258 (258-AAACCG-259 changed to 258TCG ACG-259) and an engineered PstI site at codon His-293
(293-CATGAGTAA changed to 293-CTGCAGTAA). Fusions
to the fdnI gene were made at engineered PstI sites at
codons Arg-15 (14-GATCGCGCC-16 changed to 14-GACT
GCAGC-16), Met-52 (52-ATGGGACGC-54 changed to ATC
TGCAGC-54), Gly-112 (111-GCCGGGCAA-113 changed to
111-GCCTGCAGA-113) and Leu-153 (152-CTGCTG-153
changed to 152-CTGCAG-153), and at an engineered HindIII
site at codon Gly-216 (215-GAAGGGATA-217 changed to
215-GAAGCTTTA-217). Each F(fdn–lacZ ) gene (translational) fusion was constructed by isolating fdn fragments that
extend from an introduced EcoRI site located 313 nucleotides
upstream of the fdnG transcription initiation site (Li and
Stewart, 1992) to each of the newly introduced downstream
restriction sites. Fragments were cloned into vectors pNM480
or pNM482 (Minton, 1984) as appropriate to establish an in
frame fdn–lacZ fusion junction (at lacZ codon Val-10). Thus,
fusions to fdnH include an upstream fdnG + gene, and fusions
to fdnI include upstream fdnG + and fdnH + genes. All fusions
(except those to codon Lys-44) include a change of fdnG
codon 196 from UGA (Sec) to UCA (Ser) to ensure that UGA
decoding to selenocysteine did not limit fdnGHI expression
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
Topological analysis of Tat substrates
(Berg et al., 1991b). Each fusion junction includes up to 10
additional codons derived from the pNM48x polylinker.
Each F(fdn–phoA) gene fusion was constructed by cloning
a ¢phoA–kan cassette into the engineered site in each of
the F(fdn–lacZ) plasmids described above. The cassettes
PHOK2 or PHOK3 (Rodríguez-Quiñones et al., 1994) were
used as appropriate to establish the reading frame. Fusions
were to phoA codons Val-28 (PHOK3) and Ala-33 (PHOK2).
Each fdn–phoA fusion junction includes up to 25 additional
codons derived from the PHOK linker.
The F(fdnG–bla)K44 and F(fdnG–cat)K44 gene fusions
were constructed from the EcoRI–BamHI (codon 45) fdnG ¢
fragments described above. The ¢bla and ¢cat genes were
obtained by PCR amplification from plasmids pBR322
(source of bla from transposon Tn3) and pACYC184 (source
of cat from transposon Tn9) respectively. The primers used
for amplification introduced in frame BamHI sites just
upstream of the bla and cat initiation codons (5¢-N NNN
ATG-3¢ changed to 5¢-G GAT CCG-3¢) and HindIII sites just
downstream of the termination codons. Primers were: 5¢-GAA
ggatccGAGTATTCAACATTTCC-3¢, bla 5¢ primer (restriction
site in lower case); 5¢-ATGAGTaagcttGGTCTGACAGTTA
C-3¢, bla-3¢ primer (termination codon underlined); 5¢-GAAGC
ggatccGGAGAAAAAAATCACTG-3¢, cat 5¢ primer; 5¢-TTAAA
aagcttACGCCCCGCCCTGCC-3¢, cat-3¢ primer. The EcoRI–
fdnG¢–BamHI–¢bla–HindIII and EcoRI–fdnG¢–BamHI–¢cat–
HindIII fusions were assembled in the pUC19-derived
polylinker of plasmid pK19 (Pridmore, 1987). The BamHI–
¢bla–HindIII and BamHI–¢cat–HindIII cassettes were also
cloned into the native BamHI site at fdnG codon Asp-792
to generate the F(fdnG–bla)D792 and F(fdnG–cat)D792 gene
fusions.
The F(sufI-x)A27 fusions contain DNA encoding the SufI
signal sequence up to and including the Ala-Ser-Ala cleavage site. In most cases, the constructs contained 503 bp
upstream of sufI including 429 bp of the upstream plsC gene.
For the F(sufI–lacZ)A27 fusion, only 128 bp upstream of the
sufI initiation codon (54 bp of plsC sequence) were included.
The F(sufI–bla)A27 fusion was constructed as follows. The
DNA coding for the signal sequence of SufI and upstream
regions was amplified with primers 5¢-GCGCtctagaCTTC
GGGCAGTTGTACTGGTTAACC-3¢ and 5¢-GCGCgatatcTGC
GCTGGCCTTCAGGGGAAC-3¢, digested with XbaI and
EcoRV and cloned into pBluescript-KS II to give plasmid
pHH15. The b-lactamase gene lacking DNA encoding the
native signal peptide was amplified using primers 5¢-GCGC
ctcgagCACCCAGAAACGCTGG-3¢ and 5¢-GCGCgggccc
TTACCAATGCTTAATCAGTG-3¢ (stop codon underlined),
digested with XhoI and ApaI and cloned into pHH15 to give
plasmid pHH16. As the F(sufI–bla)A27 construct is present in
pBluescript-KS II, it was necessary to subclone the fusion into
a vector that did not carry an additional copy of the b-lactamase gene. Therefore, pHH16 was digested with ApaI, end
filled with T4 polymerase and then digested with XbaI to
excise the fusion region. This was ligated into pDHB5700 that
had been digested with Ecl136I and XbaI to give plasmid
pHH18. For the construction of F(sufI–phoA)A27, DNA coding
for the signal sequence of SufI and upstream regions was
amplified with primers 5¢-GCGCtctagaCTTCGGGCAGTTG
TACTGGTTAACC-3¢ and 5¢-GCGCggatccGCTGCGCTGGC
CTTCAGG-3¢. The amplified product was digested with XbaI
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1005–1021
1017
and BamHI and cloned into pSK4158, which had been
digested previously with the same enzymes, to give plasmid
pHH21. For the construction of F(sufI–lacZ)A27, DNA encoding the SufI signal sequence and 128 bp of upstream
DNA was amplified by PCR using the following primers
5¢-GCGCgaattcCGCCGAGCTCGATAAAGAAGTCGC-3¢ and
5¢-GGCggatccCCGGCTGCGCTGGCCT TCAGGG-3¢, with
MC4100 DNA as template. The product was digested with
EcoRI and BamHI and cloned into pVJS2213 digested
with the same enzymes to give plasmid pHH26.
Construction of strains by transduction using phage P1
followed the methods of Miller (1992).
Biochemical methods
Subcellular fractions were prepared as described previously
(Sargent et al., 1998; Stanley et al., 2000). The DMSO reductase activity of DmsAB in subcellular fractions is normally
quite labile but, in our hands, can be significantly stabilized
by a combination of anoxic conditions, the use of Trisbased buffers and the addition of a protease inhibitor cocktail (Roche). Fractionation efficiency was monitored using
acid phosphatase and glucose-6-phosphate dehydrogenase
as periplasmic and cytoplasmic marker enzymes respectively (Atlung et al., 1989; Sargent et al., 1998). Only data
from fractionations in which the marker enzyme activities
were ≥95% correctly localized are reported. b-Lactamase
activity was measured spectrophotometrically by following
the hydrolysis of 7-(thienyl-2-acetamido)-3-[2-(4-N,Ndimethylaminopheylazo)pyridinium-methyl]-3-cephem-4 carboxylic acid (Calbiochem). b-Galactosidase activity was
assessed by the hydrolysis of ONPG (Sigma) using the
method described by Miller (1992). Chloramphenicol acetyltransferase activity was determined according to the method
of Shaw (1975) by reaction of the Coenzyme-A product with
5,5¢-dithiobis-(2-nitrobenzoic acid). Alkaline phosphatase
activity was measured spectrophotometrically by following
the hydrolysis of p-nitrophenyl phosphate. The activity of
DMSO reductase was measured spectrophotometrically as
described previously (Silvestro et al., 1988) using benzyl
viologen (BV) radical as electron donor and TMAO as electron acceptor.
SDS–PAGE used the buffer system described by
Laemmli (1970). Antigens were detected after immunoblotting using either the Protoblot (Promega) or ECL (AmershamPharmacia Biotech) detection systems. Antiserum against
FDH-N (Graham and Boxer, 1981) was kindly provided by
Professor D. Boxer, University of Dundee, UK. Polyclonal
antisera directed against TEM b-lactamase, chloramphenicol
acetyltransferase, alkaline phosphatase and b-galactosidase
were obtained from 5 PRIME Æ 3 PRIME.
Acknowledgements
We thank Professor D. H. Boxer for anti-FDH-N antiserum
and partially purified FDH-N. V.S. thanks Professor Peter
Hinkle for educational discussions. Work in Norwich was
supported by the Biotechnology and Biological Sciences
Research Council through project grant 88/P09634 and by
the CEC through grant QLK3-CT-1999. Work in Ithaca was
1018
N. R. Stanley et al.
supported by US Public Health Service grant GM36877 from
the National Institute of General Medical Sciences. N.R.S.
was the recipient of a Norwich Research Park Studentship.
F.S and T.P. are Royal Society University Research Fellows.
B.C.B. is R. J. P. Williams Senior Research Fellow at
Wadham College, Oxford.
Note added in proof
The structure of E. coli FDH-N has now been determined by
X-ray crystallography (Jormakka et al., in press). The FdnG
and FdnH subunits are located at the face of the transmembrane FdnI protein that, on the basis of marker fusion analysis in this work and in Benoit et al. (1998), has been assigned
a periplasmic location. The FDH-N structure thus confirms
the periplasmic localization of FdnG that is deduced herein.
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