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INTRODUCTION |
Until recently, protein translocation in bacteria was thought to
take place either by the sec pathway or by specialized
translocation systems (1). The translocation of proteins by the
sec pathway requires the protein to remain in an open
conformation during the process of translocation and extensive
information exits for sec-mediated translocation (2, 3). The
sec system has also been implicated in the translocation of
selected periplasmic membrane-extrinsic and membrane-intrinsic
proteins. For example, leader peptidase is targeted by the
sec pathway (4), and a newly described protein, YidC (a
component of sec-apparatus), was shown to target
specifically membrane-intrinsic proteins (5-7). We and others (8-11)
have reported an alternative protein targeting and translocation system termed membrane targeting and translocation
(Mtt),1 also termed twin
arginine translocase. The Mtt system is comprised of at least three
proteins, MttA1A2B (also called TatABC) and has
been shown to transport fully folded proteins to or across the membrane
(8-10, 12). Unlike the sec system, no known integral membrane protein is targeted by the Mtt pathway (13). The discovery of
the Mtt system explained the translocation into the periplasm of
cofactor-containing proteins that were assembled in the cytoplasmic compartment. These proteins have a long N-terminal leader with a
conserved twin arginine motif ((S/T)RRXF(X/L)K)
(11). They have diversity in subunit composition, molecular weight, the
nature of the redox cofactors, and cellular localization (13-15).
Bacterial proteins utilizing the Mtt pathway include some proteins
without cofactors such as SufI, a member of the copper oxidase family, fusion proteins containing a reporter gene (e.g.
-lactamase), and even malfolded proteins (9, 16-19). Components of
the Mtt protein translocation machinery are found in at least half of the complete bacterial genomes available to date as well as the genomes
of chloroplasts and plant mitochondria. However, Mtt is totally absent
from animal genomes (8, 11, 13).
Dimethyl sulfoxide (Me2SO) reductase is encoded by the
dms operon and is expressed under anaerobic growth
conditions in the absence of nitrate (20, 21). Extensive investigation
has shown that the DmsAB subunits form a membrane extrinsic dimer
facing the cytoplasm, and DmsC, an integral membrane protein, serves as
a membrane anchor (22). Recently, we have confirmed that DmsA has an
N-terminal twin arginine leader that functions as a membrane-targeting
signal and is also essential for the stability of the holoenzyme
(15).
Membrane targeting via the Mtt system is not limited to
Me2SO reductase. Formate dehydrogenase-O is targeted to the
membrane (but not translocated across) by a twin arginine leader (23). More recently, chlorophenol reductive dehalogenase (encoded by cprAB genes) of Desulfitobacterium
dehalogenase was shown to be a membrane-bound enzyme, apparently
targeted to the membrane by the twin arginine leader. CprA undergoes
processing resulting in cleavage of the leader, and the mature form of
the enzyme was localized to the cytoplasmic face of the membrane with
the CprB subunit presumably serving a membrane anchor function (24). Thus certain membrane-bound enzymes whose active sites face the cytoplasm appear to be targeted to the membrane by the Mtt system (15,
23, 24).
To better understand the mechanism of the membrane targeting of
Me2SO reductase, we have constructed several mutant strains defective in the mtt genes. We provide experimental evidence
to show that Mtt catalyzes the targeting of DmsAB to the cytoplasmic face of the membrane, but not its translocation, and that a functional Mtt system is required for processing of DmsA to the mature form and
for the stability of the DmsAB dimer.
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EXPERIMENTAL PROCEDURES |
Materials--
Molecular biology reagents and ECL Western
blotting detection kit were purchased from Life Technologies, Inc., and
Amersham Pharmacia Biotech, respectively. Oligonucleotides were
obtained from the Department of Biochemistry DNA core facility,
University of Alberta, Edmonton, Canada. DNA polymerases
(Taq and Elongase enzyme mix) were purchased from Life
Technologies, Inc. Pfu DNA polymerase was purchased from
Stratagene. All other reagents used were of the highest purity
available commercially.
Methods--
Media, growth conditions, anaerobic growth profiles
on minimal media composed of glycerol as a carbon source and
Me2SO, trimethylamine N-oxide (TMAO), or
fumarate as the terminal electron acceptor were as described previously
(15, 25). Procedures for restriction enzyme digestions, agarose gel
electrophoresis, elution of DNA fragments from agarose gels, and
separation of proteins using SDS-gel electrophoresis, phage P1
transduction, construction of deletion strains using the method of
homologous recombination were carried out by standard procedures (25,
26). Osmotic shock methods for preparing the periplasmic fraction and
preparation of soluble and everted membrane vesicles by French press
lysis were as described earlier (15, 27). Protein was estimated by a
modification of the Lowry procedure (28). Electrophoresis of proteins
was carried out in 7.5 or 10% SDS-polyacrylamide gels, and
proteins were transferred to nitrocellulose filters for Western blotting analysis using antibodies to the DmsA, DmsB, and
-lactamase proteins (22, 29). Me2SO reductase activity was monitored by the oxidation of reduced benzyl viologen by the substrate TMAO. Fumarate reductase activity was monitored in an assay system similar to
the Me2SO reductase, except fumarate was used as a
substrate (22, 30).
Bacterial Strains and Plasmids--
Bacterial strains and
plasmids used are described in Table I.
Construction of plasmids pDMS190 and pDMS160 carrying the entire dms operon under the control of the lac or the
native dms promoter, respectively, has been described
previously (15, 31).
Construction of Plasmids--
Procedures for the cloning of the
entire mtt operon
(mttA1A2BC)
into the vectors pMS119EH (AmpR), pTZ19R
(AmpR), or pBR322 (TetR) and cloning of the
mttB gene into pMS119EH were as described below and in the
legend to Table II. The relevant
mtt gene sequences were amplified using PCR and chromosomal
DNA from a wild-type strain (HB101) as a template with the commercially
supplied buffers (Life Technologies, Inc.) and Taq,
Pfu, or Elongase DNA polymerases in a thermocycler unit
programmed for optimum temperatures for annealing and extension for the
individual gene amplification reactions (32).
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Table II
List of oligonucleotides used and the derived plasmids
The oligonucleotides used and the derived plasmids are indicated. The
oligonucleotide pairs used in the polymerase chain reaction to generate
the expression plasmids, the deletion plasmids, or the CAT cartridge
are as indicated.
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The entire mtt operon was amplified using the
oligonucleotide pairs RAT20/GZ-01 (see Table II for oligonucleotide
sequences) with flanking EcoRI and SalI
restriction enzyme sites at the 5' and 3' ends, respectively. The PCR
product (2544 bp) was cloned into the EcoRI and
SalI sites of pMS119EH or pTZ19R to yield the plasmids
pMTT(AmpR) and pMTT/19R (AmpR), respectively.
These plasmids carry an 82-bp (5' end) and a 32-bp (3' end) flanking
sequence outside of the mttABC. The plasmid carrying the
mttA1A2BC
sequences under control of the native mtt promoter were
amplified by PCR using the oligonucleotide pairs GZ-08/GZ-07 with an
engineered EcoRI site at the 5' end. The PCR DNA (3596 bp)
was digested with EcoRI and PstI enzymes,
respectively, to yield 3375 bp of the mtt gene sequence. The
PstI site is present within the 3' end of the amplified DNA,
outside of the mtt operon. The
EcoRI/PstI fragment was cloned into
EcoRI/PstI-cleaved pBR322 resulting in deletion
of the bulk of the ampicillin coding region of the vector to yield the
plasmid pMTT-322 (TetR). This plasmid also differs from
pMTT or pMTT/19R in that it has an extended 5' region (588 bp upstream
of the ATG start codon of MttA1) and 3' sequences (356 bp)
downstream of the mtt operon. Plasmid pMTTB gene
was amplified by PCR using the oligonucleotides GZ-04/RAT22. The PCR
DNA (827 bp) was cut with EcoRI and SalI prior to
cloning into pMS119EH to generate the plasmid pMTTB.
Plasmid pDMS193 encodes the DmsAB dimer under control of the
dms promoter and was constructed using PCR with primers
DS-23/DS-24 and pDMS160 as template DNA to amplify a 188-bp DNA
fragment that covers the 3' end of dmsB with an engineered
stop codon and flanking SstI and
SalI/XhoI restriction enzyme sites at the 5' and
3' ends, respectively. The plasmid pDMS190 was digested with
SstI and SalI to remove part of 3' end of
dmsB and the entire dmsC (1073 bp). The DNA was
gel-eluted, and the larger fragment (7494 bp) containing the entire
dmsA and most of dmsB was mixed with the PCR DNA
and ligated to yield the plasmid, pDMS193.
Deletion or Insertional Inactivation of the mtt Genes--
The
mttA2 gene was inactivated by insertional
mutagenesis. The plasmid pMTT/19R was cut with SacII at the
unique site within mttA2. The recessed ends were
filled in to create blunt ends. A blunt-ended chloramphenicol
acetyltransferase (CmR) gene cartridge, CAT, generated
using PCR (see below) was inserted at the filled in SacII
site of the pMTT/19R plasmid to generate p
MTTA2/CAT. The
CmR gene sequence (CAT) was amplified using pACYC184 DNA as
template and the oligonucleotides CAT5/CAT3 (Table II). The CAT
cartridge has BamHI-flanking ends and was also used to
generate various mtt gene deletions as summarized below and
in Table II.
Construction of Deletion Strains--
The mtt mutant
plasmids p
MTT/CAT, p
MTTA1/CAT,
p
MTTA2/CAT, p
MTTB/CAT, p
MTTC/CAT were constructed
as summarized in Table II. The mutant plasmids were introduced
individually into the recombination deficient strains JC7623 or D308 as
described earlier (25, 33) by homologous recombination. The appropriate
deletions from the rec
strains were P1
transduced into the recipient strain, TG1 to generate DSS640
(
mtt), DSS641 (
mttA1), DSS642
(
mttA2), DSS643 (
mttB), and
DSS644 (
mttC), respectively. Strains deleted in the
chromosomal dms (
dmsABC) and the
mtt operons (
mttABC) were constructed by P1
transduction of the
mttABC mutation (p
MTT/CAT),
mttB (p
MTTB/CAT), or
mttC (p
MTTC/CAT)
into DSS301 (
dms) resulting in strains DSS740, DSS743,
and DSS744, respectively. Strain K-38/pGP1-2 was P1-transduced using
the phage lysates from DSS640 carrying a mtt operon deletion
to generate K-38/pGP1-2/
mtt.
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RESULTS |
Characterization of mtt Deletion Strains--
Deletions
in mttA2, mttB, and
mttA1A2BC
failed to support anaerobic growth on GD (15, 25) (Fig.
1, A, C, and D) or
GT (15, 25) minimal medium (data not shown). Deletion of
mttA1 showed moderate inhibition of growth
compared with the wild-type strain, TG1 (Fig. 1A). This was
shown to be due to a compensating effect of a functional homologue of
MttA1, TatE, present on Escherichia coli
chromosome (9). Deletion of mttC alone had very little effect on the growth profiles measured under our experimental conditions (Fig. 1B). Growth on glycerol/Me2SO
medium was restored when the Mtt polypeptides were expressed from a
multicopy plasmid pMTT carrying the entire mtt operon in the
mtt strain (Fig. 1C). Similarly, growth of the
mttB strain was corrected by a plasmid (pMTTB) expressing
the MttB polypeptide (Fig. 1D). The defective growth
phenotype observed for the strain deleted in
mttA2 was also corrected by a plasmid expressing
the Mtt polypeptides (data not shown). These results clearly
demonstrate that the mttA2 and mttB
genes are critical for anaerobic respiration on Me2SO.

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Fig. 1.
Growth of the mtt
deletions on glycerol/Me2SO minimal medium.
Bacterial growth was monitored at 37 °C in a Klett
spectrophotometer. Klett units are plotted as a function of time
(hours) for the various strains tested. For a description of the
strains see Table I. A, TG1 versus DSS641
( mttA1) and DSS642
( mttA2). B, TG1 versus
DSS644 ( mttC). C, TG1 versus DSS640
( mttA1A2BC)
and DSS640/pMTT. D, TG1 versus DSS643
( mttB) and complementation of
DSS643/pMTTB.
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As a control for the growth experiments, we investigated the growth of
our mtt mutants on GF (15, 25) minimal medium. None
of the fumarate reductase polypeptides bear a twin arginine leader, and
this enzyme is not targeted to the membrane by the Mtt system, thus
growth on GF medium should be unaffected in these mutants.
Surprisingly,
mttA1 and
mttA2 showed limited growth, whereas
mttB and a total deletion of the mtt operon
(
mtt) nearly abolished growth on GF medium (Fig.
2, A and B).
Anaerobic growth on GF medium was restored by in vivo
complementation of the
mttB strain by pMTTB (Fig.
2B); similarly the
mtt and
mttA2 strains were complemented by expression
plasmids carrying the entire mtt operon (data not shown).
The mtt deletions grew normally under aerobic conditions and
anaerobically with nitrate (data not shown).

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Fig. 2.
Anaerobic growth of E. coli
on glycerol/fumarate minimal medium was measured as described in
Fig. 1. A, TG1 versus DSS641
( mttA1) and DSS642
( mttA2). B, TG1 versus
DSS640
( mttA1A2BC),
DSS643 ( mttB), and DSS643/pMTTB.
C, DSS301 ( dms) versus
the double mutants, DSS740 ( dms, mtt),
DSS743 ( dms, mttB), and
DSS740/pDMS160.
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We hypothesized that the lack of growth on GF medium resulted from the
insertion of DmsC into the membrane without the DmsAB subunits.
Previously, we showed that expression of DmsC in the absence of DmsAB
is lethal (34). In confirmation of this hypothesis growth was observed
on GF medium for the double mutants DSS740 and DSS743
(
mtt
dms and
mttB
dms, respectively) (Fig. 2C) which was similar
to the parent strain TG1 (Fig. 2A) or a
dms (DSS301) strain (Fig. 2C). Predictably, introduction of the
DmsABC expression plasmid (pDMS160) into the double mutant,
mtt
dms (DSS740), suppressed growth on
fumarate (Fig. 2C), whereas the expression of the DmsAB
subunits without DmsC (pDMS193) did not suppress growth (data not shown).
Comparison of Me2SO Reductase Activities in the Control
and the mtt Deletion Strains--
To correlate growth to the membrane
association of the reductase activity, we measured enzyme activities
with the artificial electron donor, benzyl viologen with TMAO as a
substrate. As expected, the control strain TG1 showed up to 94% of the
activity in the membrane fraction (Table
III). The reductase activity in the
mutants DSS640 (
mtt), DSS642
(
mttA2), and DSS643 (
mttB) was
predominantly soluble. Complementation of the deletions with
appropriate plasmids resulted in membrane-bound reductase activity.
mttA1 (DSS641) and
mttC
(DSS644) exhibited profiles close to the control strain. The
distribution and the specific activity of fumarate reductase remained
unaffected indicating that this anaerobic enzyme was not influenced by
the Mtt pathway (data not shown).
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Table III
Comparison of the Me2SO reductase activity in control and
mtt deletion strains
Bacterial cultures were grown anaerobically in peptone-fumarate medium
for 24 h. Preparation of membrane (M) and soluble (S, periplasm plus
cytoplasm) fractions and the assay of the Me2SO reductase using
benzyl viologen (BV) and TMAO as the electron donor-acceptor species
were as described under "Experimental Procedures." Total activity
units represent the combined activities of membrane and soluble
fractions. The percent distribution and the specific activity of the
individual fractions for each bacterial strain tested were as
indicated. TG1, DSS640
( mttA1A2BC), DSS641
( mttA1), DSS642
( mttA2), and DSS643 deletions strains
are described under "Experimental Procedures."
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Stability of Me2SO and TMAO Reductases in the mtt
Deletions--
The total Me2SO reductase activity in
mttA2 and
mttB was greatly
reduced, compared with the control, DSS641 and DSS644 (Fig. 3A). By comparison the
periplasmic enzyme TMAO reductase was stable in these mutants even
though it accumulated in the cytoplasm (Fig. 3B). We used
DSS301 as the strain for comparison of TMAO reductase activity because
this strain has a deletion in the dms operon and thus
Me2SO reductase does not interfere with the benzyl
viologen-TMAO assay. Upon in vivo complementation, the
specific activity as well as the total activity of Me2SO
reductase approached the control values.

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Fig. 3.
Comparison of the stabilities of TMAO and
Me2SO reductases in the mtt
deletions. A, bacteria were grown in
peptone/fumarate medium (TG1, DSS640, DSS641, DSS642, DSS643, and
DSS644) or B, peptone/fumarate/TMAO medium (DSS301, DSS740,
and DSS744). Membrane and supernatant (periplasm + cytoplasm) fractions
were prepared and assayed for enzyme activity as described under
"Experimental Procedures," using TMAO as substrate for both TMAO
and Me2SO reductases. The combined total activity units in
the membrane and supernatant fractions were used for the
histogram.
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Localization of DmsAB--
As a precursor to examining the
processing of the DmsA leader, we felt that it was essential to
re-examine the cytoplasmic localization of DmsAB. Eliminating the
anchor DmsC should simplify interpretation as expression of DmsAB
closely mirrors a typical dual subunit Mtt substrate such as the
E. coli hydrogenase-2. Such an experiment was reported
earlier, with a construct that encoded DmsAB and the first two
transmembrane loops of DmsC (pDMSC59X) (8). Expression from this
construct showed some accumulation of soluble DmsAB dimer in the
periplasmic compartment (8) leading us to propose that DmsC served a
"stop-transfer" role. However, as the experiment lacked adequate
controls for lysis and the quality of the periplasmic fraction and to
eliminate potential problems arising from expression of a partial DmsC
subunit, we constructed a plasmid encoding only DmsAB (pDMS193).
Localization was studied in the control strain (DSS301) and the
mtt deletions by immunoblotting of appropriate fractions
(Fig. 4). As an internal control, the plasmid encoded
-lactamase was also monitored in these same
fractions loaded identically on a separate gel and transferred onto a
blot for probing with
-lactamase-specific antibody (Fig. 4).
-Lactamase is a known sec-dependent and
periplasmically localized enzyme. The DmsAB subunits encoded by the
plasmid pDMS193 in the control strain (DSS301) were predominantly
cytoplasmic. The membrane fraction had a very small amount of DmsAB,
presumably due to the contamination from occluded soluble reductase in
the membrane pellet. The periplasmic fraction was almost devoid of
DmsAB. In these samples
-lactamase was predominantly in the
periplasmic fraction. A small amount of the
-lactamase was noted in
the cytoplasmic fraction and none in the membrane fraction. These
findings clearly indicate that the DmsAB subunits are present only in
the cytoplasm. A soluble cytoplasmic enzyme marker (glucose-6-phosphate
dehydrogenase) was also monitored in these fractions (35).
Glucose-6-phosphate dehydrogenase was present only in the cytoplasmic
fraction, also validating the quality of the extracts prepared (data
not shown). As expected, the DmsAB subunits were localized to the
cytoplasmic fraction in the
mtt strain. The
-lactamase
(Fig. 4) and the glucose-6-phosphate dehydrogenase (data not shown)
were present in the expected cell compartments.
-Lactamase export
was not affected by deletion of the mtt genes. These
experiments were carried out in a strain totally lacking DmsC
(DSS301/pDMS193) and a strain with sub-stoichiometric levels of DmsC
relative to DmsAB (DSS640/pDMS193). However, it is expected that in
strain DSS640/pDMS193 the DmsAB dimer would accumulate in the
cytoplasm, regardless of the level of the DmsC expression from the
chromosomal copy of the dms operon.

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Fig. 4.
Localization of the DmsAB and
-lactamase proteins in the various cellular
fractions by immunoblot analysis. Preparation of the membrane
(M), cytoplasmic (C), and periplasmic
(P) fractions from cells expressing the DmsAB dimer and the
plasmid-encoded -lactamase (pDMS193) were as described under
"Experimental Procedures." The strains tested, DSS301
( dms) and DSS640 ( mtt), are indicated. The
immunoreactive DmsA and DmsB subunits in the various fractions were
compared with an external standard (Std) of purified DmsAB.
The left top and bottom panels indicate the
analysis for the Me2SO reductase, and the right
top and bottom panels illustrate the immunoblot
analysis for the -lactamase.
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Processing of the Twin Arginine Leader Sequence of DmsA in the mtt
Deletions--
The DmsA leader sequence is critical for expression,
membrane targeting, and stability of the reductase (15). We examined the processing of DmsA by immunoblot analysis in whole cell lysates derived from the mtt deletions (Fig.
5). Whole cell lysates closely reflect
the levels of the reductase under various conditions of growth and
expression and minimize artifactual degradation resulting from
preparation of membrane and soluble fractions. DSS301/pDMS190 and
DSS644/pDMS190 showed good processing with the majority of DmsA
migrating at a position corresponding to the mature form based on the
mobility of the purified mature protein (lane with Me2SO
reductase standard). A very faint slower migrating band was also
observed in these strains corresponding to the precursor form of DmsA.
On the other hand, DSS640/pDMS190 and DSS643/pDMS190 showed
predominantly the precursor form of DmsA. The expression levels of DmsA
were similar or slightly higher in all the strains tested, indicating
that the mtt deletions did not impair the expression of the
reductase. The presence of only the precursor form of the reductase in
DSS640 and DSS643 confirms that at a minimum MttA2 and MttB
are required for processing of the reductase.

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Fig. 5.
Immunoblot analysis of the DmsA subunit.
An immunoblot analysis of the lysates prepared from the whole cells
harboring the entire dms operon (pDMS190, top
panel) or the strains expressing only the DmsAB dimer (pDMS193,
lower panel) were examined. The individual bacterial strains
used for the preparation of the whole cell lysates were identified
above each sample lane. The immunoreactive DmsA bands were
identified as precursor (p), mature (m), and
degraded (d) forms. Std refers to the purified
Me2SO reductase used to compare the relative mobilities of
the various species of DmsA identified.
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DISCUSSION |
Considerable information has accumulated on the role of Mtt in the
translocation of periplasmic proteins. The necessity for MttA1, MttA2, and MttB but not MttC has been
documented for TorA, Fdn-N, Hya 1, SufI, and YacK proteins (9, 19, 36).
Me2SO reductase is a membrane-bound protein with the
extrinsic DmsAB subunits facing the cytoplasm (22). Assembly of
Me2SO reductase requires Mtt, and DmsA has a typical twin
arginine leader that is essential (15). It was of interest to see if
Me2SO reductase had the same requirements for Mtt proteins.
We constructed a series of mtt chromosomal deletions and
confirmed that Me2SO reductase requires MttA2
and MttB using anaerobic growth measurements (Fig. 1). Deletion of
MttA1 had only a marginal effect on growth. This is due to
the redundancy of MttA1 resulting from the presence of the
homologous gene, ybec (tatE), on the E. coli chromosome. A strain deleted for tatA
(mttA1) and tatE failed to direct the Me2SO reductase to the membrane implying a role for these
proteins (9). MttC was not required, and MttC has been shown to be a protein exhibiting nuclease activity and is apparently unrelated to the
Mtt system (37). We have noted that amplification of Me2SO
reductase in an mttC deletion strain showed only 50% of the
membrane-bound reductase compared with the wild type. We assume that in the mttC deletion, the mtt mRNA may
be less stable resulting in the lower activity measurements.
Complementation of the mutants with appropriate mtt genes on
plasmids corrected the phenotype. However, we have observed a
significant lag in the complemented strains (Fig. 1).
Overexpression of the Mtt proteins from multicopy plasmids hinders
growth and in some instances may totally inhibit cell growth (36).
These studies confirm and extend the observations of others (9, 36) who
have shown that the growth defects on Me2SO and TMAO are
due to a generalized defect in the Mtt system.
As a control for the growth studies, we examined the ability of the
mtt deletions to grow on TMAO, fumarate, and nitrate. TMAO
reductase is a soluble periplasmic reductase, with a twin arginine
leader, that utilizes Mtt for translocation. Growth and activity
results with this enzyme reflected the Me2SO reductase data
(data not shown). Fumarate and nitrate reductase are membrane-bound enzymes that lack twin arginine leaders and do not utilize Mtt. We were
surprised to find that the mtt deletions (
mtt
and
mttB) failed to grow on fumarate (Fig. 2) but grew
normally on nitrate (data not shown). By use of a double deletion for
both dms and mtt, we traced the phenotype to the
expression of incorrectly assembled Me2SO reductase in the
mtt deletions. Expression of DmsC, the Me2SO
reductase anchor subunit in the absence of correctly assembled DmsAB
catalytic dimer is lethal (34). As expected, deletion of both the
dms operon and the mtt operon restored growth on
fumarate. As nitrate represses the expression of dmsABC,
growth on nitrate was unaffected.
The enzyme activity distribution of Me2SO reductase (Table
III) and TMAO reductase (9) also confirms that the proteins are mislocalized in strains with deletions of the mtt operon.
Me2SO reductase was very labile compared with TMAO
reductase in these mutants (Fig. 3) suggesting rapid proteolytic
degradation of mistargeted DmsAB.
A large amount of experimental evidence utilizing immunoblotting of
periplasmic fractions obtained by osmotic shock and chloroform washing,
protease susceptibility, lactoperoxidase-catalyzed iodination, immunogold electron microscopy of everted membrane preparations, TnphoA fusions, and electron paramagnetic resonance
monitoring the effects of the probe dysprosium(III) on an engineered
3Fe4S cluster in DmsB indicated that DmsAB faces the cytoplasm (22, 38). Others (11) have suggested that DmsAB faces the periplasm based
solely on the presence of a twin arginine leader sequence in DmsA. In
this study we have re-examined the question of the localization of
DmsAB in both wild-type and mtt deletions (Fig. 4). Our
results once again confirm that DmsAB is cytoplasmic.
In a recent study, the localization of DmsAB was compared in a strain
expressing a partial DmsC subunit (pDMSC59X) with full-length DmsC
originating from the chromosomal copy of the dms operon. In
those experiments we reported (8) some DmsAB in the periplasm in the
absence of full-length DmsC, leading us to suggest that DmsC might
serve a stop-transfer role. In the current experiments, with a complete
DmsC deletion, DmsAB subunits were only observed in the cytoplasm. It
is possible that the absence of DmsC, or sub-stoichiometric levels of
this polypeptide, could alter the localization of the DmsAB dimer.
However, the large body of experimental evidence gathered to date
supports the cytoplasmic localization of DmsAB in the wild-type and in
the reductase-amplified strains (with stoichiometric amounts of DmsC)
(22, 38) as well as DmsC-deficient strains (Fig. 5). The earlier
studies lacked compartment-specific marker enzyme controls for the
fractionation protocols, and as a result a small amount of cell lysis
and/or an increase in membrane leakiness due to expression of the
partial DmsC subunit could have contributed to the observed DmsAB in
the periplasm (8).
In the absence of the DmsC anchor subunit, one might expect that DmsAB
should be translocated to the periplasm directed by the twin arginine
leader. Why is DmsAB not translocated even in the absence of DmsC? It
could be that the DmsA leader contains some stop-transfer information.
This seems unlikely because in a previous study we showed that
replacement of the dms leader with the tor leader
did not affect the topology of DmsAB (15). It appears that DmsAB
contains information in the mature polypeptides that prevents
translocation to the periplasm. This conundrum is not limited to DmsAB.
The localizations of FdoGH in E. coli (23) and CprAB in
D. dehalogenase (24) were also shown to be cytoplasmic despite the presence of twin arginine leader sequences. Furthermore, N-acetylmuramyl-L-alanine amidase (AmiA), a twin
arginine leader sequence containing protein, was shown to be targeted
and translocated by an Mtt-independent mechanism (39). These studies
imply that information in the total polypeptide and not solely the
signal sequence determines the localization of a given protein. Elegant information on the importance of information in the mature protein for
membrane targeting came from studies of a TorA-leader peptidase (TorA-Lep) fusion protein. The full-length TorA-Lep fusion protein was
directed to the membrane via the Sec pathway and not by the Mtt system,
even though a twin arginine leader was present (4). Conversely, the
DmsA twin arginine leader was shown to mediate the export of a fusion
protein (yeast cytochrome c) via the Mtt system (40). Taken
together, these results favor the view that both the twin arginine
leader and the mature polypeptide contribute to the final localization
of a specific protein.
The results of whole cell immunoblotting (Fig. 5) clearly indicate that
the MttA1, MttA2, and MttB proteins are
required for processing of the DmsA leader. This was seen with both the
catalytic DmsAB dimer and the holoenzyme indicating that the DmsC
anchor does not play a role in processing of pre-DmsA to mature DmsA. It is not clear how the DmsA leader is processed. There is no evidence
to date that any of the known Mtt subunits are directly involved in the
processing of the leader sequence. It has been assumed that the leader
peptide is cleaved by leader peptidase I which faces the periplasm (4).
Attempts to study the processing of the DmsA leader using a conditional
lethal leader peptidase mutant were inconclusive, due to the poor
growth of this strain and the difficulty in obtaining total peptidase
deficiency under our experimental conditions (data not shown). Since
none of the substrates of the Mtt system studied to date are true
soluble cytoplasmic proteins, the observation of apparent DmsA
processing in the cytoplasm should be interpreted with caution. The
DmsAB dimer attached to the membrane-bound Mtt system possesses a
leader sequence of 45 amino acids that is sufficient to traverse the bilayer and mediate processing on the periplasmic side, without the
bulk of the DmsAB ever crossing the bilayer. The processed DmsAB would
then lack affinity for the Mtt system and could be released into the
cytoplasm. Alternatively, the DmsA leader could be cleaved by a novel
cytoplasmic protease. Interestingly, mutation of the two conserved
alanines to asparagines at the DmsA leader cleavage site did not affect
the membrane targeting or the processing (15).