genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of ...

27 Pages·2008·1.22 MB·English

by  Matthew Delisa

JBC Papers in Press. Published on September 25, 2008 as Manuscript M802660200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M802660200
GENETIC TOGGLING OF ALKALINE PHOSPHATASE FOLDING REVEALS SIGNAL
PEPTIDES FOR ALL MAJOR MODES OF TRANSPORT ACROSS THE INNER MEMBRANE
OF BACTERIA
Matthew J. Marrichi1, Luis Camacho2*, David G. Russell2 and Matthew P. DeLisa1,3
From School of Chemical and Biomolecular Engineering1, Department of Microbiology and
Immunology, College of Veterinary Medicine2 and Department of Biomedical Engineering3,
Cornell University, Ithaca, NY 14853 USA
Running Head: Export pathway specificity revealed by PhoA fusions
Address correspondence to: Matthew P. DeLisa, 254 Olin Hall, Ithaca, NY 14853; Phone: [phone];
Fax: [phone]; Email: [email protected]
*Current address: Novartis Institute for Tropical Diseases, 10 Biopolis Road #05-01 Chromos, Singapore

Prediction of export pathway specificity in Despite recent advances in bioinformatic
prokaryotes based on sequence information analysis of N-terminal protein export signals (1-
alone remains a challenging endeavor owing to 6), a priori prediction of export pathway
the similar overall architecture of N-terminal specificity based on sequence information alone
signal peptides for the Sec-, SRP- and Tat- remains a challenging endeavor complicated by
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dependent export pathways. Thus, we sought the similar overall architecture of N-terminal o
w
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to create an experimental strategy for facile, export signals (Fig. 1a). Since bioinformatic lo
a
unbiased discovery of pathway specificity tools for predicting pathway specificity typically de
d
conferred by N-terminal signals. Using a rely on experimentally confirmed signal fro
m
limited collection of E. coli strains that allow peptides, these approaches can be biased toward h
protein oxidation in the cytoplasm or, predicting substrates with signal sequences ttp
conversely, disable protein oxidation in the whose primary structure does not differ greatly ://w
w
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periplasm, we were able to discriminate the from those of the learning set. The TnphoA .jb
specific mode of export for alkaline transposon probe developed by Manoil and c.o
phosphatase (PhoA) fusions to signal peptides Beckwith (7) has been an extraordinarily useful brg/
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for all the major modes of transport across the experimental tool for verifying and discovering g
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inner membrane (Sec, SRP or Tat). Based on signal peptides in numerous bacterial species on es
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these findings, we developed a mini-Tn5 phoA a genome-wide scale (8,9). TnphoA is a n
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approach to isolate export pathway-specific derivative of the Tn5 transposon that enables the ec
e
signals from libraries of random fusions generation of protein fusions to Escherichia coli m
b
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between exported proteins and the phoA gene. alkaline phosphatase (PhoA; EC3.1.3.1) devoid r 2
3
Interestingly, we observed that reduced PhoA of its native amino-terminal export signal. The , 2
0
was exported in a Tat-independent manner resulting chimeric proteins only confer 18
when targeted for Tat secretion in the absence phosphate hydrolase activity to cells if the
of the essential transolcon component TatC. protein fusion is capable of export out of the
This suggests that initial docking to TatC cytoplasm. Consequently, TnphoA can be used
serves as a key specificity determinant for Tat- to analyze proteins localized to the periplasm,
specific routing of PhoA and in its absence, inner and outer membranes or extracellularly.
substrates can be re-routed to the Sec pathway The ability to assay living cells for PhoA
provided they remain compatible with the Sec activity is facilitated by the use of the
export mechanism. Finally, the utility of our chromogenic substrate 5-bromo-4-chloro-3-
approach was demonstrated by experimental indolyl phosphate (XP) or by selective growth
verification that 4 secreted proteins from on a medium containing a sole carbon or
Mycobacterium tuberculosis carrying putative phosphate source that requires PhoA hydrolysis
Tat signals are in fact bona fide Tat substrates to be metabolized (10). The general utility of
and thus represent potential Tat-dependent this genetic construct is exemplified by the large
virulence factors in this important human number of applications reporting its use
pathogen. including, for example, identification of cell
surface and secreted virulence factors (11-16),
1
Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
dissection of membrane protein topology (17- colony (34,35). This approach enabled
20), determination of sites within proteins that discrimination between cells expressing PhoA
are permissive to large insertions without fusions with cleavable signal peptides (mostly
disrupting function (21) and epitope tagging of Sec substrates) from those that spanned the inner
proteins at internal positions (8,21). membrane (mostly SRP substrates) but did not
In general, the TnphoA strategy is very shed light on the specific targeting route. A
efficient at uncovering proteins that carry an N- further shortcoming of the TnphoA system is its
terminal Sec signal peptide. This is because (i) limited ability to detect export via the twin-
PhoA itself is a native Sec-dependent substrate arginine translocation (Tat) pathway, whose
and (ii) the majority of proteins that carry an N- substrates contain unique Arg-Arg signal
terminal signal peptide for transport across the peptides and are known to fold in the cytoplasm
cytoplasmic membrane of Gram-negative prior to transiting the inner membrane (36-38).
bacteria or the cell wall of Gram-positive For instance, in a recent genome-wide screen for
bacteria are substrates of the Sec pathway N-terminal signal peptides in Psuedomonas
(22,23). The Sec pathway consists of the aeruginosa, 310 PhoA fusions were identified of
SecYEG translocase formed by the SecYEG which only 1 (RnfG, PA3493) was predicted to
integral membrane proteins and a molecular be a Tat substrate (39) despite the fact that as
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motor, SecA, which drives translocation of many as 57 Tat-dependent substrates have been o
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unfolded substrates in an ATP-dependent predicted for this organism (4). The reason for lo
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manner (22-24). The bulk of proteins targeted to this is that the bacterial Tat transporter accepts de
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SecYEG are routed in a post-translational only those proteins that have attained a native or fro
m
manner with assistance from the dedicated near-native structure in the cytoplasm (40-42). h
molecular chaperone SecB (25,26); although for Since PhoA folding is dependent upon the ttp
some substrates, including PhoA, post- catalytic formation of disulfide bonds that can ://w
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translational export can proceed without only form in the periplasm (43), PhoA fusions .jb
assistance from SecB (27). Alternatively, a are incapable of transiting the Tat pathway in c.o
subset of exported proteins are routed co- wildtype bacteria (41). However, PhoA can be brg/
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translationally following their recognition as exported by the Tat pathway under specific g
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ribosome-bound nascent chains by the signal conditions, such as when the cytoplasm is es
t o
recognition particle (SRP) (28,29) that directs rendered more oxidizing by deletion of the trxB n
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substrates to the FtsY receptor (30) and gor genes that encode the two major cytoplasmic ec
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ultimately the SecYEG translocase. This reductases in E. coli (41). m
b
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mechanism is responsible for the export of both In this study, we sought to expand the r 2
3
soluble periplasmic proteins (31,32) and, with utility of TnphoA screening for (i) resolving the , 2
0
assistance from YidC, integral membrane targeting specificity (e.g., SecB-independent vs. 18
proteins (29,33). SecB-dependent vs. SRP-dependent) of exported
Importantly, while the use of TnphoA in proteins and (ii) detecting export via the Tat
bacteria is capable of detecting genes that pathway. Using mini-Tn5 phoA-generated
encode proteins exported via the Sec and SRP libraries in combination with a limited collection
pathways, it does not provide sufficient of isogenic E. coli strains derived from strain
resolution to distinguish between these different DR473 that allow toggling of PhoA folding, we
modes of SecYEG targeting. To address this have isolated PhoA fusions for all major modes
limitation, previous studies explored the use of of inner membrane transport in E. coli.
TnphoA in combination with the leaky outer Additionally, we have employed this strategy to
membrane of E. coli carrying the lpp-5508 experimentally verify that 4 secreted proteins
mutation such that free periplasmic PhoA from Mycobacterium tuberculosis carrying
diffused away from cells and hydrolyzed XP in putative Tat signal peptides are bona fide Tat
the surrounding medium to yield a “blue halo” substrates and thus represent potential Tat-
around the colony while membrane-bound PhoA dependent virulence factors in this important
could only hydrolyze intracellular XP and the human pathogen.
resulting blue color was localized within the
2
Construction of the plasmid pBR322-
Experimental Procedures Gm was carried out by replacement of the Tcr
Bacterial strains and plasmids. The strains cassette in pBR322 with the gentamycin
and plasmids used in this study are listed in resistance gene. pBR322-Gm was then
Table 3. E. coli strains were routinely grown in converted into a destination vector with an
LB medium (Difco) broth at 30ºC or 37ºC with insertion of the Gateway RfC.1 cassette
antibiotics added at the following (Invitrogen) into the SspI/ScaI sites, creating the
concentrations: 100 µg/mL ampicillin; 20 vector pBRGC.1. Plasmid pVV16 (45) was
µg/mL chloramphenicol; 10 µg/mL gentamycin; converted into a destination vector named pVV-
50 µg/mL hygromycin; 50 µg/mL kanamycin; GC.1-FH by insertion of a modified version of
50 µg/mL spectinomycin. M. smegmatis strains the Gateway RfC.1 cassette containing a Flag
affinity epitope at the 3’ end into the
were grown in Middlebrook 7H9 medium
NdeI/HindIII sites. All derivatives of pVV-
(Difco) supplemented with 0.2% glycerol and
GC.1-FH and pBRGC.1 were created using
0.05% Tween 80; L-Lysine was added at a
Gateway cloning technology according to
concentration of 80 µg/mL for the strains
manufacturer’s protocols (Invitrogen). The
MB692, PM759 and JM578, and all cells were
plasmids pCueO-AP and pSufI-AP were
grown at 37ºC with antibiotics added at the same D
constructed by first inserting the 1,412-bp E. coli o
concentrations as listed above. M. smegmatis w
n
cells were also grown on solid Middlebrook phoA gene with a modified 5’ end to include a loa
7H10 media (Difco) supplemented as indicated mini multi-cloning site (NheI, PsiI and XhoI ded
above but with 40 µg/mL of L-Lysine when sites) into the HindIII/SalI sites of pBR322, from
necessarGy.e netic disruption of DHB4 and DR473 yeniecloddiningg vEe.c tcoorl i pcMueCOS -oΔrs sEP.h cooAli. s uTfIh ewna, s DPNCAR http://w
cells was achieved using P1vir transducing amplified and inserted in the NheI/PsiI sites w
w
phage. Briefly, donor cells containing the gene pMCS-ΔssPhoA. Plasmid pssMBP-PhoA was .jb
c
of interest with a selectable marker were grown constructed by PCR amplification of the first .org
in LB broth at 37°C overnight and subcultered 143-bp of malE from pMalE and insertion of the by/
resulting product into the HindIII/XhoI sites of g
into fresh media containing 5 mM CaCl , 10 u
2 e
mdiMlut ioMng aSnOd4 alalnodw e0d. 2t%o ggroluwc ofsoer 1a.t5 ah 1a0t 03-7f°oCld. pcoMnCstSru-cΔtsesdP hboyA in. sePrltaisnmg iadn p8M64C-bSp- ΔPsCsBRl apCro dwuacst st on D
e
corresponding to M. tuberculosis blaC lacking c
Lytic phage was then added and the culture was em
the first 93-bp but with a 33-bp Flag epitope tag b
grown until “clearing” occurred, upon which e
added to the 3’ end into the SpeI/EcoRI sites of r 2
time the phage was harvested through 3
pSALect. To construct plasmid pVV-ssAg85A- , 2
centrifugation and stored at 4°C with 01
BlaC-FH, a 120-bp PCR product encoding the 8
chloroform. Recipient DHB4 or DR473 cells
M. tuberculosis Ag85A signal peptide was first
were grown in appropriate antibiotics at 37°C
cloned into the SalI/SpeI sites of pMCS-
overnight and centrifuged at 5,000 x g for 3 min.
ΔssBlaC. Then, a 14-bp ribosome binding site
Cells were then resuspended in fresh media
was appended to the 5’ end of the DNA
containing 5 mM CaCl , 10 mM MgSO and
2 4 encoding Ag85A-BlaC via PCR and the entire
0.2% glucose and lytic phage carrying the
1010-bp construct was cloned into the
marked gene of interest was added at a 10-fold
NdeI/HindIII sites of pVV16. This same
dilution. Cells and phage were grown at 37°C
procedure was repeated to create pVV-ssAg85C-
for 30 min, subsequently supplemented with 100
BlaC-FH, pVV-ssModD-BlaC-FH and pVV-
mM sodium citrate, grown for one more hour
ssPepA-BlaC-FH by inserting DNA encoding
and plated on LB agar containing the appropriate
the signal peptides (72-bp, 117-bp, and 96-bp,
antibiotics and 100 mM sodium citrate
respectively) inserted at the 5’ end of ΔssBlaC.
overnight. Cells containing the genetic
Plasmid pBR-BamHI-Gm was constructed by
disruption were recovered and the resistance
inserting the BamHI restriction site into the
marker was removed using the pCP20 helper
SspI/PstI sites of pBR322-Gm.
plasmid as previously described (44).
3
Subcellular fractionation. Cytoplasmic and mixed, and spotted in 40 µL aliquots onto a
periplasmic fractions were prepared from cells nitrocellulose membrane on LB agar and grown
that had been induced for protein expression at at 30ºC for 16 h. The membrane was then
30ºC for 8 h in the presence of either arabinose resuspended in 10 mL of fresh LB and used to
or glucose as indicated. Following protein inoculate 200 mL of LB supplemented with 300
induction, cells were pelleted by centrifugation µg/mL kanamycin and 10 µg/mL gentamycin,
at 3,000 x g for 15 min at 4ºC and then subjected followed by growth at 37ºC for 16 h. Upon
to the ice-cold osmotic shock procedure as conjugation, approximately 104 recipient cells
previously described (46). The quality of all containing both the marked transposon and the
fractionations was determined by expression vector of interest were selected and
immunodetection of the cytoplasmic GroEL pooled together. Plasmid DNA was then
protein (41). extracted from these cells and used to transform
Western blot analysis. Proteins were separated competent DR473 cells that were grown on LB
by SDS-PAGE and Western blotting was agar supplemented with appropriate antibiotics,
performed as described previously (47). Briefly, 50 µg/mL XP, and either 0.2% glucose or 0.2%
all lanes of sodium dodecyl sulfate (SDS)-12% arabinose at 30ºC for two days. For each
polyacrylamide gels (Bio-Rad) were loaded with library, 12 colonies that exhibited a strong blue
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samples prepared from an equivalent number of phenotype were restreaked onto the same media ow
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cells harvested for each experiment. The and grown at 30°C for two days to confirm the lo
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following primary antibodies were used: phenotype. Plasmid DNA was then prepared ded
monoclonal mouse anti-PhoA (Sigma-Aldrich) from all positive clones and sequenced using a fro
m
diluted 1:20,000; monoclonal mouse anti-Flag primer specific for the antisense strand of the 5’ h
(Stratagene) diluted 1:3,000 and polyclonal anti- ttp
GroEL (Sigma-Aldrich) diluted 1:10,000. end of the phoA gene. ://w
Construction and screening of genomic w
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Secondary antibodies were either goat anti- library. Genomic DNA was isolated from .jb
mouse (Promega) or goat anti-rabbit (Promega) MC4100 cells and partially digested using c.o
diluted 1:2,500. Following development of blots Sau3AI as previously described (39). Fragments brg/
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using the Immun-Star HRP Substrate Kit (Bio- between 0.75-3.0 kb were excised from a 1% g
u
Rad) and visualized using X-ray film (Kodak), agarose-Tris-borate-EDTA gel and gel purified est o
membranes were stripped in solution consisting n
(QIAGEN), then ligated into the BamHI site of D
of 2.0% SDS, 7.0% β-mercaptoethanol, 0.03% pBR-BamHI-Gm. This library of genomic DNA ece
m
NaCl and 0.0025% Tris, reblocked and probed was then electroporated into competent MC4100 b
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with anti-GroEL antibody. cells and conjugated with SM10 λ-pir cells r 23
Monitoring of PhoA activity in intact cells. E. carrying pUTphoA as described above. The , 20
1
coli cells were grown in LB supplemented with resulting library, comprised of transposon 8
appropriate antibiotics at 37ºC overnight and
insertions into genomic DNA, was isolated and
streaked onto LB agar supplemented with
electroporated into competent DR473 cells.
appropriate antibiotics, 50 µg/mL 5-bromo-4- Screening of the library was performed on BCIP
chloro-3-indolyl phosphate (XP, Sigma- indicator plates supplemented with either
Aldrich), and 0.2% arabinose or 0.2% glucose glucose or arabinose as described above. Cells
and grown at 30ºC for two days. Streaks that displaying a bright blue phenotype were
attained a blue phenotype were classified as reconfirmed, after which their plasmid DNA was
export competent while cells that appeared isolated and sequenced using a primer specific
white/colorless were classified as incapable of for the antisense strand of the 5’ end of the phoA
PhoA export from the cytoplasm. gene.
Isolation of mini-Tn5 phoA insertions. SM10 Bioinformatic prediction of Tat substrates.
λ-pir cells carrying pUTphoA and MC4100 cells Analysis was carried out as previously described
carrying the expression vector of interest were (3). Briefly, a hidden Markov model (HMM)
grown overnight at 37ºC in LB supplemented was developed using a training set constructed
with appropriate antibiotics. Cells were then from experimentally confirmed Tat substrates
pelleted, washed three times with fresh LB,
4
present in E. coli and P. aeruginosa. The Following introduction of the dsbA::kan allele
previously developed HMM for Tat motifs using into DR473, protein oxidation in the periplasm
hmmbuild (http://hmmer.wustl.edu/) was is impaired and the resulting strain is designated
calibrated with hmmcalibrate and used to search either C:red/P:red or C:ox/P:red depending on
the annotated proteins from the chromosome of whether arabinose or glucose respectively, is
M. tuberculosis H37Rv (GenBank accession supplemented (41). Finally, DR473 derivatives
number NC_000962.2) with hmmsearch. Signal carrying an insertional deletion in the gene
peptides were restricted to fall within the first 50 encoding the SecB chaperone or the essential
amino acids of the given protein sequence and Tat translocase component TatC were used to
all putative signals were crosschecked using inactivate specific targeting routes.
SignalP 3.0 (1) and TatP 1.0 (2). We hypothesized that the use of these
Antimicrobial susceptibility testing. M. different strains and growth conditions in
smegmatis cells were grown for two days at combination with the redox-dependent folding
37°C in Middlebrook 7H9 medium and behavior of PhoA (41,43) would reveal targeting
appropriate antibiotics as outline above. specificity of different signal peptide-PhoA
Cultures were then diluted 1000-fold in fresh fusions as shown schematically in Figure 1b. For
medium and 5 µL were pipetted onto LB agar instance, we reasoned that in C:red/P:ox cells,
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supplemented with 0.2% glycerol and 75 µg/mL PhoA with its native export signal (X = PhoA in ow
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carbenicillin (Sigma). Cells were then grown at Fig. 1b) would be efficiently exported in a post- lo
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37°C for four days and growth was determined translational, SecB-independent manner where ded
by single-colony formation within spots. rapid oxidation by DsbA in the periplasm would fro
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catalyze formation of the two disulfide bonds h
that are critical for the stability and catalytic ttp
Results activity of the protein (43) (Fig. 1b). To test this, ://w
PhoA-based screen for discriminating export w
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pathway specificity. First, we sought to develop we monitored in vivo export of native PhoA in .jb
an experimental strategy using a Tn5-based C:red/P:ox and C:red/P:red cells grown on LB c.o
phoA derivative for facile, unbiased discovery of agar plates supplemented with arabinose and brg/
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pathway specificity conferred by N-terminal XP, a colorimetric substrate that emits a visible g
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signals. To accomplish this, we exploited a blue color when hydrolyzed by PhoA. Since XP est o
diffusion through the inner membrane of E. coli n
limited collection of E. coli mutant strains that D
allow protein oxidation in the cytoplasm or, is very inefficient (our unpublished observations ece
and see below), it is an excellent indicator of m
conversely, disable protein oxidation in the b
e
periplasm (41). Specifically, strain DR473 PhoA activity in the E. coli periplasm or r 2
3
(Table 3) used in this study carries deletions in extracellular medium. As expected, the , 20
the genes trxB (thioredoxin reductase) and gor C:red/P:ox cells exhibited a strong blue 18
phenotype (Fig. 2a), while the C:red/P:red cells
(glutaredoxin reductase) such that the cytoplasm
exhibited a colorless phenotype (Fig. 2a),
of these cells favors the oxidation of protein
consistent with the known DsbA-dependence of
thiols (41,48). In addition, these cells lack phoA
PhoA folding and activity (49). To confirm that
and have a chromosomal copy of the trxB gene
export of PhoA proceeded in a SecB-
under control of an arabinose-inducible
independent manner (50), when the same
promoter such that the addition of arabinose or
construct was expressed in C:red/C:ox secB-
glucose can be used to effectively toggle the
cells the blue phenotype remained (Fig. 2a). For
cytoplasm as a reducing or oxidizing
comparison, we assayed a fusion between
environment, respectively. In the presence of
mature PhoA and the 26-residue signal peptide
arabinose, this strain background is designated
derived from the SecB-dependent substrate
C:red/P:ox because the cytoplasm is relatively
maltose binding protein (MBP, encoded by the
reducing while the periplasm is oxidizing. On
malE gene) (27). Indeed, SecB dependence was
the contrary, in the presence of glucose, the
observed as the expression of ssMBP-PhoA in
strain is designated C:ox/P:ox as both the
C:red/C:ox cells elicited a strong blue phenotype
cytoplasm and periplasm are oxidizing.
5
that was completely abolished in cells lacking export of ssDsbA-PhoA would effectively
SecB (Fig. 2a). bypass the cytoplasm and, as a result, the
Since native PhoA does not require the chimera would be unaffected by the cytoplasmic
anti-folding activity of SecB prior to export, we redox status. In agreement with this notion, both
hypothesized that PhoA might fold prematurely C:red/P:ox and C:ox/P:ox cells expressing
in C:ox cells and fail to be exported. Indeed, ssDsbA-PhoA appeared blue when grown on XP
expression of native PhoA in C:ox/P:ox cells indicator plates (Fig. 2a). As expected for SRP-
resulted in colonies that appeared colorless, dependent export, the blue phenotype was
suggesting that the C:ox environment rendered unaffected by the absence of SecB or TatC, but
PhoA translocation-incompetent. An identical deletion of DsbA resulted in the complete
colorless phenotype was observed in C:ox/P:ox absence of blue coloration (Fig. 2a).
secB- cells (data not shown), confirming that We next sought to develop our PhoA-
premature folding of PhoA and not SecB based screen for verification of Tat-dependent
binding was the cause of translocation protein export. As mentioned above, export of
incompetence in the C:ox environment. On the PhoA by the Tat pathway is observed only in
contrary, we observed that C:ox/P:ox cells strains that enable oxidative protein folding in
expressing ssMBP-PhoA produced a strong blue the cytoplasm (41). This was corroborated by
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phenotype (Fig. 2a), suggesting that binding of the observation that expression of fusions o
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ssMBP-PhoA by SecB is sufficient to maintain between mature PhoA and Tat-specific signal lo
a
the protein in an export-competent conformation peptides derived from either E. coli CueO or de
d
even in an oxidizing folding environment. As SufI (ssCueO-PhoA or ssSufI-PhoA, fro
m
expected for Sec-dependent targeting signals, respectively) in C:ox/P:ox cells conferred a blue h
expression and localization of native PhoA and phenotype (Fig. 2b). In a reduced, incorrectly ttp
ssMBP-PhoA was unaffected by a deletion in folded state, PhoA is unable to be translocated ://w
w
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tatC (Fig. 2a) that is known to completely because, with few exceptions, the Tat pathway .jb
abolish export via the Tat export pathway (51). appears to export only native or native-like c.o
In all of the above cases, Western blot analysis proteins (40,41,52). Indeed, C:red/P:ox cells brg/
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was used to confirm that the phenotypic data expressing the same constructs resulted in a g
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correctly reported the subcellular localization of colorless phenotype, consistent with the fact that es
t o
each PhoA fusion (shown in Fig. 3a for native reduced PhoA is incompetent for Tat export n
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PhoA). As a control, mature PhoA lacking a (41). Unlike the SRP- or Sec-dependent PhoA ec
e
signal peptide expressed in C:red/P:ox and fusions, expression of the ssCueO-PhoA and m
b
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C:ox/P:ox cells resulted in a colorless phenotype ssSufI-PhoA constructs in C:ox/P:red dsbA- r 2
3
(data not shown), indicating lack of export and cells resulted in a blue phenotype (Fig. 2b), , 2
0
confirming that the XP indicator was incapable indicating that oxidative folding of Tat-targeted 18
of diffusing into the cytoplasm under the PhoA occurs in the cytoplasm and is not
conditions tested here. Western blot analysis of dependent on DsbA for folding or activity (41).
subcellular fractions generated from these same As expected, neither ssCueO-PhoA nor ssSufI-
cells confirmed the cytoplasmic location for PhoA were localized to the periplasm in tatC-
mature PhoA irrespective of the cytoplasmic cells (Fig. 2b). Unexpectedly, in C:red/P:ox
redox status (Fig. 3b). tatC- cells both ssCueO-PhoA and ssSufI-PhoA
To further explore the ability of our were localized to the periplasm in an active
PhoA-based screen to discriminate signal conformation (Fig. 2b). This result suggests that
peptides based on their specific mode of export, in the absence of TatC, which has been shown to
we evaluated the phenotype of cells expressing a be the primary recognition component for Tat
chimera between the E. coli DsbA signal peptide signal peptides (53), reduced PhoA is exported
(ssDsbA) and PhoA. It was previously reported in a Tat-independent manner, perhaps via the
that ssDsbA is capable of routing passenger Sec pathway as suggested in previous studies
proteins such as E. coli thioredoxin-1 to the co- (41,54). However, this promiscuity was never
translational SRP-dependent pathway (32). observed when TatC was present or when PhoA
Thus, we hypothesized that co-translational was expressed under C:ox conditions,
6
suggesting that both TatC and the folded state the phoA gene using mini-Tn5 phoA and
are specificity determinants in Tat-targeted screening the resulting DNA library in
PhoA export (see below for more details). C:ox/P:ox tatC- cells. According to Table 1, this
A transposon-based strategy for isolating combination of strain and growth conditions was
export pathway-specific PhoA fusions. more stringent than that used above as it could
Collectively, the above data reveal a logic table only produce SecB- or SRP-dependent export
for PhoA pathway specificity in DR473 and its signals. Upon screening, we selected 12 blue
derivatives grown in the presence of arabinose colonies at random and found that each of these
or glucose (Table 1; see also Fig. 1b). Based on clones expressed an identical fusion comprised
this logic, we hypothesized that judicious of the first 86 amino acids of DsbA fused in-
selection of strain and growth conditions could frame to the N-terminus of mature PhoA
be employed to isolate signal peptides according (ssDsbA86-PhoA). To verify whether this was
to their specific mode of inner membrane export. an SRP-dependent export signal, we screened
To test this notion, a library of random fusions this clone in cells lacking SecB and found that,
of the gene encoding the SecB-dependent unlike ssMalE372-PhoA, the ssDsbA86-PhoA
substrate MBP and the phoA gene were fusion conferred a blue phenotype to C:ox/P:ox
generated by mini-Tn5 phoA insertions (55). For secB- cells grown on glucose (data not shown).
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mini-Tn5 phoA-generated hybrid proteins to Western blot analysis further confirmed that o
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have high enzymatic activity, the phoA gene ssDsbA86-PhoA localized in the periplasm lo
a
must be fused to a target gene coding for a regardless of the cytoplasmic oxidation state of de
d
signal that promotes protein export (7,55). To DR473 cells (Fig. 4b). As expected for an SRP fro
m
increase our chances of success, we ‘relaxed’ the export signal, the absence of a functional Tat h
screening stringency by expressing the MBP- pathway did not prevent translocation of ttp
PhoA hybrids in C:ox/P:ox cells, a combination ssDsbA86-PhoA to the periplasm (Fig. 4b). ://w
w
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of host and growth conditions that can These experiments revealed the potential to .jb
potentially yield signals for SecB-, SRP- and isolate SecB-independent signal peptides from c.o
Tat-dependent export (see Table 1). Following transposon libraries. brg/
y
screening of cells on XP indicator plates Isolation of Tat-dependent PhoA fusions. As g
u
supplemented with glucose, 12 blue colonies a final test of our transposon screening es
t o
were isolated at random, each of which encoded approach, we sought to isolate signal peptides n
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an in-frame fusion between the first 372 amino for the Tat pathway. This represented a ec
e
acids of MBP and the N-terminus of TnphoA significant challenge because Tat export signals m
b
e
(ssMBP372-PhoA). Because library screening in have largely been precluded from TnphoA-based r 2
3
C:ox/P:ox cells can produce clones directing screens (e.g., ref.(39), an observation that is best , 2
0
SecB-, SRP- or Tat-specific export, we further explained by the fact that PhoA is unable to 18
tested ssMBP372-PhoA in C:ox/P:ox cells obtain a correctly folded, Tat export-competent
lacking SecB or TatC and observed a colorless conformation in the cytoplasm of wildtype
phenotype and blue phenotype, respectively bacteria (41). However, as seen above,
(data not shown). Furthermore, Western blot C:ox/P:ox cells can efficiently translocate
analysis confirmed that ssMBP372-PhoA was fusions between Tat signal peptides and mature
localized in the periplasm when expressed in PhoA in a Tat-specific manner. Encouraged by
either C:ox or C:red cells (Fig. 4a). Collectively, this finding, we used mini-Tn5 phoA to generate
these experiments revealed the potential of the two libraries of random fusions between the
assay to isolate SecB-dependent substrates from gene encoding either CueO or SufI and the phoA
transposon libraries. gene and screened each of these libraries in
To further explore the ability of our Tn- C:ox/P:ox cells. Two unique clones were
based strategy to isolate signal peptides based on isolated, namely ssCueO34-PhoA and
pathway specificity, we next sought to identify ssSufI178-PhoA. As mentioned above,
SRP-dependent export signals from a transposon screening C:ox/P:ox cells can yield SecB-, SRP-
library. This was achieved by generating random and Tat-dependent export. Therefore, we tested
fusions between the gene encoding DsbA and ssCueO34-PhoA and ssSufI178-PhoA in the
7
C:ox/P:red genotype which can be used to easily PhoA, whereas C:red/P:ox tatB- and tatA/E-
discriminate between Sec/SRP and Tat signals cells were incapable of localizing ssSufI-PhoA
(see Table 1). Consistent with Tat-dependent to the periplasm. For comparison, when the
export, both ssCueO34-PhoA and ssSufI178- same Tat-deficient strains were grown on
PhoA conferred a blue phenotype to C:ox/P:red glucose (C:ox), no detectable PhoA export was
cells (data not shown). Western blot analysis of observed (Fig. 5b), highlighting the fact that
subcellular fractions confirmed that these clones only reduced, and not oxidized, PhoA is capable
were localized to the periplasm only when TatC of Tat-independent export. To determine
was present (Fig. 5a). In C:red/P:ox cells, whether the observed Tat-independent export
ssCueO34-PhoA and ssSufI178-PhoA were not was due to the lack of specificity cues that might
exported (Fig. 5a). Interestingly, as was seen be contained within the mature region of SufI,
above for ssCueO-PhoA and ssSufI-PhoA, Tat- we created an in-frame fusion between the entire
independent export was observed for these two coding region of sufI and the sequence encoding
clones when each was expressed in cells with a the mature region of PhoA. As shown in Fig. 5c,
reducing cytoplasm (C:red) that also lacked C:red/P:ox cells expressing SufI-PhoA exhibited
TatC (Fig. 5a). Though ssCueO and ssSufI have the same Tat-independent export as seen above
been shown to display some pathway for ssSufI-PhoA. Thus, taken together our
D
promiscuity (41,54), the specific contribution of results suggest that Tat targeting specificity is o
w
n
TatC and of the folding state to this promiscuous maintained by the signal peptide binding site lo
a
export had not previously been reported. This formed by TatC, the folding state of PhoA de
d
phenomenon was explored in greater detail (oxidized vs. reduced) and the N-terminal signal fro
m
below. peptide but does not appear to be effected by h
Tat pathway specificity is maintained by the regions within the mature SufI protein. ttp
substrate binding component TatC. Our rather Genome-wide identification of E. coli export ://w
w
w
surprising observation that Tat-targeted PhoA signals specific for a given pathway. To .jb
constructs were exported in a Tat-independent determine whether the method holds promise for c.o
manner when both (i) PhoA was reduced and (ii) genome-wide, unbiased screening of signal brg/
y
TatC was absent from cells, is likely due to the sequences specific for a given pathway, we g
u
fact that TatC is the initial docking site for constructed a library of random E. coli DNA es
t o
ssCueO-PhoA and ssSufI-PhoA and in its fragments fused to TnphoA. DR473 cells were n
D
absence, both of these substrates can be re- transformed with this library and plated on ec
e
routed to the Sec pathway provided they are in a arabinose (C:red/P:ox) such that the cytoplasm m
b
e
Sec export-competent conformation (i.e., was reducing. Recall that DR473 cells with a r 2
3
reduced). If TatC were indeed the docking site, reducing cytoplasm allow for identification of , 2
0
we reasoned that Tat-independent export of Sec substrates exported in either a SecB- 18
ssCueO-PhoA and ssSufI-PhoA, presumably via dependent or independent manner as well as
the Sec pathway, would only be observed in SRP substrates, but not Tat substrates (Fig. 1b
tatC- cells. In contrast, Tat deficient mutants and Table 1). Using these screening conditions,
lacking TatB or TatA/E would not be expected we identified five in-frame fusions to PhoA that
to exhibit Tat-independent export of ssCueO- were able to promote export across the inner
PhoA and ssSufI-PhoA because, despite the membrane as indicated by a strong blue
inability of these cells to complete the entire Tat phenotype when cells were grown on BCIP
export process, the presence of the TatC indicator plates. The five identified genes were:
component would ensure initial targeting of (i) atoS, (ii) ydjX, (iii) yfhR, (iv) yjgX, and (v)
PhoA to the Tat pathway (53,56,57). Recently, ymgD. Consistent with the screening conditions,
in vivo genetic studies have yielded suppressor all five hits were predicted to be either integral
mutants of TatC that permit export of non- membrane proteins (atoS, ydjX, yfhR, and yjgX)
canonical Tat signals, further underscoring its according to the TMHMM membrane protein
role in signal peptide docking (58,59). As seen topology prediction method of Sonnhammer and
in Figure 5b, growth of C:red/P:ox tatC- cells coworkers (61) or to carry N-terminal Sec export
resulted in Tat-independent export of ssSufI- signals (ymgD) according to the signal peptide
8
prediction tool SignalP 3.0 (1). As expected, no independently confirmed to localize PhoA out of
Tat substrates were identified. Moreover, atoS the cytoplasm (data not shown).
and ydjX were previously shown to encode for Discovery of novel M. tuberculosis Tat export
integral inner membrane proteins with two and signals. Based on our ability to detect E. coli
five transmembrane helices (60). In contrast, the export signals, we next sought to determine
ymgD clone, which does not contain any whether our assay could be used to
transmembrane helices, was determined to be a experimentally verify Tat signal peptides from
SecB-indepenent substrate according to BCIP other bacterial hosts. Due to the important role
screening of secB+ and secB- cells expressing that protein secretion systems, including the Tat
the YmgD-PhoA fusion (Fig. 6a). Importantly, export pathway, play in the virulence of
all five clones were independently confirmed to bacterial pathogens (64,65), we set out to
localize PhoA out of the cytoplasm by identify Tat-targeting sequences from the
blue/white screening and Western blot analysis genome of the Gram-positive human pathogen
(data not shown). M. tuberculosis using our PhoA-based screening
The same DR473 cell library was also system. Both the M. tuberculosis and M.
screened on glucose (C:ox/P:ox), which we smegmatis genomes contain open reading frames
expected would yield signals for SecB- with homology to components of the Tat export
D
dependent, SRP-dependent and Tat-dependent system (TatABC) and the Tat system is o
w
n
export (Fig. 1b and Table 1). However, SecB- apparently essential in M. tuberculosis (66,67) lo
a
independent translocation of PhoA protein (and also Camacho, Russell, DeLisa, S. Ehrt and de
d
fusions would escape detection under these D. Schnappinger, unpublished observations). fro
m
conditions. Upon screening, four in-frame However, to date, no more than five Tat- h
fusions to PhoA were identified that dependent substrates have been reported for ttp
corresponded to: (i) narG, (ii) wzc, (iii) ybhR, mycobacteria (66,68,69) despite the fact that the ://w
w
w
and (iv) yqjF. As expected, three of these hits genomes of M. smegmatis and M. tuberculosis .jb
are inner membrane proteins: wzc encodes a are predicted to encode 49 (69) and, as c.o
membrane-bound protein-tyrosine kinase with determined own bioinformatic analysis, 61 brg/
y
two transmembrane helices (62), ybhR encodes (Supplementary Table 1) putative Tat substrates, g
u
an ABC transporter with six membrane helices respectively. It is noteworthy that several of the es
t o
(60) and yqjF is a quinol oxidase subunit with M. tuberculosis Tat substrates predicted by our n
D
four transmembrane helices (60). The fourth analysis have been experimentally confirmed as ec
e
clone, narG, encodes the α-subunit of nitrate authentic Tat substrates, including BlaC, PlcA, mb
e
reductase A (NarGHI) that was previously PlcB and Rv2525c (66,68). Alignment of the r 2
3
shown to be exported out of the cytoplasm via entire set of M. tuberculosis substrates and , 2
0
the Tat pathway (63). Indeed, NarG was found comparison of amino acid frequencies reveals a 18
to export PhoA in a Tat-dependent manner (Fig. prototypical Tat motif followed immediately by
6b). Interestingly, it did not exhibit the same a moderately hydrophobic alanine-rich h-region
promiscuous export as was seen for all the other (Fig. 7a). Of the 61 predicted Tat substrates in
Tat signals expressed in C:red/P:ox tatC- cells M. tuberculosis, prior proteomic analysis
(Fig. 6b). It is noteworthy that this protein identified 4 of these, namely Rv0125 (PepA),
possesses only the vestige of a twin-arginine Rv0129c (Ag85C), Rv1860 (ModD) and
motif (MSKFLDRFRYFKQKGET…; where the Rv3804c (Ag85A), as extracellular proteins (70)
non-canonical Tat motif is underlined) (63) and, (and also Drs. Sarah Fortune and Eric Rubin,
as a result, it has escaped detection by current personal communication), suggesting that a
bioinformatic tools (54). Thus, the identification subset of secreted M. tuberculosis proteins are
of the Tat substrate NarG exemplifies the Tat substrates. We hypothesized that Tat
potential of our approach for uncovering substrates secreted to the culture medium were
unexpected, non-canonical export signals that likely candidates for virulence factors and thus
would otherwise be missed with classical opted to test these 4 candidates in our PhoA-
TnphoA-based screening or bioinformatic based screen. Using the same procedure as
analysis. As above, all four clones were outlined earlier, random fusions of the candidate
9
M. tuberculosis genes and the phoA gene were ΔtatA cells, sensitivity to carbenicillin was
generated by mini-Tn5 phoA insertions. observed (Table 2), confirming that these 4
C:ox/P:ox cells were screened and at least one proteins were authentic mycobacterial Tat
unique clone encoding an in-frame fusion substrates capable of directing Tat-dependent
between an M. tuberculosis targeting signal and export in M. smegmatis.
the N-terminus of mature PhoA was recovered
for each of the 4 candidates. Individually these Discussion
were: Ag85A217-PhoA, Ag85C312-PhoA, In the present study, we have developed an
ModD141-PhoA, and PepA121-PhoA. experimental strategy using a Tn5-based
Expression of these constructs in C:ox/P:ox transposon probe for the discovery of pathway
tatC- cells confirmed that each was a bona fide specificity conferred by N-terminal export
Tat substrate (Fig. 7b). Consistent with prior signals. Facilitated by the creation of a logic
results, export of each was blocked when the table describing PhoA export pathway
cytoplasm was rendered more reducing (Fig. specificity in DR473 cells and mutant strains
7b). As described in detail above, each of these derived thereof (see Table 1), we were able to
fusions was exported in a Tat-independent isolate export signals for all of the major modes
manner when expressed in C:red/P:ox tatC- cells of transport (Sec, SRP or Tat) across the inner
D
(Fig. 7b), indicating that this phenomenon was a membrane of E. coli. In our library screening ow
n
general feature of Tat export signals and not studies, we chose to use permissive screening lo
a
exclusive to E. coli signals. conditions such that export signals for more than de
d
Finally, to confirm that each of the one export pathway were possible. In all cases, fro
m
identified M. tuberculosis Tat signals could the true pathway specificity of each isolated h
fhaoistht,f uwlley tepsrtoemd oetaec Th aitn e ax pgoernte itnic a s meleycctoiobnac utesriniagl sciogunnatle rwscarse enth uesni nega as idlyif freerseonltv heods t bsytr aain sainmdp/oler ttp://ww
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M. smegmatis host cells (68). In this system, the altered growth conditions. However, it should be .jb
M. tuberculosis β-lactamase BlaC is used as a noted that for the discovery of SRP- and Tat- c.o
rg
reporter based on (i) the fact that it is a native M. dependent export signals, more stringent b/
y
tuberculosis Tat substrate and (ii) its ability to screening in C:ox/P:ox secB- tatC- and g
u
confer resistance to β-lactam antibiotics when C:ox/P:red cells, respectively, could be used to est o
exported in M. smegmatis. A caveat is that an M. restrict export to a single mode. We expect that n D
smegmatis strain lacking the BlaC homologue our mini-Tn5 phoA strategy could also be used ec
e
m
(BlaS) is required to eliminate the native β- to identify genes encoding inner membrane b
e
lactam antibiotic resistance of M. smegmatis proteins targeted via the SRP pathway. Indeed, r 2
3
cells. Taken together, antibiotic selection of M. initial screens for SecB-independent export , 2
0
1
smegmatis blaS- cells expressing fusions uncovered a number of PhoA fusions to the 8
between Tat signal peptides and the N-terminus tetracycline resistance protein TetA (data not
of mature BlaC provides a convenient method shown), a known inner membrane protein with
for verifying mycobacterial Tat substrates. To 12 transmembrane spans (71) that was initially
confirm the Tat-dependence of Ag85A, Ag85C, encoded in the pBR322 plasmid but removed for
ModD and PepA in mycobacteria, a fusion of this work.
the Tat signal peptide derived from each of the An interesting observation was that for
substrates and mature BlaC was expressed in M. all of the mini-Tn5 phoA libraries made using
smegmatis. Following plating on 75 µg/mL target genes (e.g., DsbA), only a single unique
carbenicillin, we observed that mc2155 ΔblaS clone was isolated in each case. While it is
currently unclear why so few unique clones were
cells carrying empty plasmid were sensitive to
uncovered, we suspect this was a result of the
carbenicillin while the same cells expressing a
relatively small size of the random libraries
fusion between ssAg85A, ssAg85C, ssModD, or
(~104 unique conjugation events) and the non-
PepA and BlaC exhibited resistance to this level
exhaustive screening of each library (only 12
of carbenicllin (Table 2). When these fusions
were expressed in Tat-deficient mc2155 ΔblaS hits were characterized from each library). A
further limiting factor is that for a given gene,
10