Download Topological characterization of the essential Escherichia coli cell

FEMS Microbiology Letters 216 (2002) 23^32
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Topological characterization of the essential Escherichia coli cell
division protein FtsW
Beatriz Lara, Juan A. Ayala
Centro de Biolog|¤a Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Cient|¤¢cas, Universidad Auto¤noma de Madrid, Cantoblanco,
28049 Madrid, Spain
Received 23 May 2002 ; received in revised form 19 July 2002; accepted 24 July 2002
First published online 30 August 2002
Abstract
The membrane topology of Escherichia coli FtsW, a 46-kDa essential protein, was analyzed using a set of 28 ftsW^alkaline phosphatase
(ftsW^phoA) and nine ftsW^L-lactamase (ftsW^bla) gene fusions obtained by in vivo and in vitro methods. The alkaline phosphatase
activities or resistance pattern of cells expressing the FtsW^PhoA or FtsW^Bla fusions confirmed only eight out of 10 transmembrane
segments predicted by computational methods. After comparison with the recent topology of Streptococcus pneumoniae FtsW, we could
identify all the fusions in absolute agreement with the predicted model: N-terminal and C-terminal ends in the cytoplasm, 10
transmembrane segments and one large loop of 67 amino acids (E240^E306) located in the periplasm.
8 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Cell division; FtsW; Lipid II; Topology
1. Introduction
The ftsW gene [1] mapped between murD and murG at
the dcw cluster was originally identi¢ed and sequenced by
Matsuhashi’s group [2]. The E. coli FtsW amino acid sequence showed a high homology to the Bacillus subtilis
SpoVE and the Escherichia coli RodA proteins that are
required for asymmetric division during sporulation and
for elongation and maintenance of the rod shape during
cell growth, respectively [3,4]. Based on these similarities,
it has been suggested that these three membrane proteins
have similar roles in cell elongation (RodA), cell division
(FtsW), and spore formation (SpoVE), respectively. Moreover, a signature pattern located in the C-terminal region
has been described for these cell cycle proteins FtsW/
RodA/SpoVE in the PROSITE database (PROSITE:
PDOC00352, PS00428) that identi¢ed 58 homologous proteins (update of January 2002).
* Corresponding author. Tel. : +34 (91) 3978083;
Fax : +34 (91) 3978087.
E-mail address : [email protected] (J.A. Ayala).
It has been predicted that FtsW might interact with
PBP3 in septum formation [1,5]. Membranes prepared
from cells overproducing both PBP3 and FtsW showed
an increased synthesis of peptidoglycan as well as of undecaprenol-linked intermediates in vitro [6]. It has been
shown that the putative enhancer function of FtsW in
the transglycosylase activity of PBP3 is not reproducible
[7]. However, the increased formation of lipid-linked intermediates suggested that FtsW might function in supply of
the lipid-linked precursor to PBP3 for the formation of the
septal peptidoglycan.
Genetic analysis of two thermosensitive alleles of the
gene (ftsW201 [8] and ftsW263 [9]) revealed that these
mutations caused a division block at an earlier stage
than that caused in strains carrying the ftsZ84 allele,
and suggested a role of FtsW at the initiation stage
of cell division. However, another allele of the gene
(ftsW1640) caused the division block later on [9], which
suggests some role of FtsW at a late step of septum formation. It has been proposed that the early function of
FtsW in cell division is the establishment of a stable FtsZ
structure, and that the late function is a link between the
events in the cytoplasm (FtsZ-ring formation) and the initiation of septal peptidoglycan biosynthesis in the periplasm [10]. By immuno£uorescence microscopy, FtsW
has been shown to be located in the middle of cell [11].
0378-1097 / 02 / $22.00 8 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 9 2 - 3
FEMSLE 10621 31-10-02
Cyaan Magenta Geel Zwart
24
B. Lara, J.A. Ayala / FEMS Microbiology Letters 216 (2002) 23^32
Septal localization of FtsW required FtsZ, FtsA, FtsQ,
and FtsL but not FtsI. Thus, FtsW is a late recruit to
the division site and is essential for subsequent recruitment
of its cognate transpeptidase FtsI (PBP3) but not for stabilization of FtsZ rings. It has been suggested that a primary function of FtsW homologs is to recruit their cognate transpeptidases to the correct subcellular location
[12].
An important step to elucidate the presumable double
function of this protein is to establish with certainty its
topology in the membrane. Based on the combined assays
of alkaline phosphatase fusion and cysteine accessibility
techniques the topology of Streptococcus pneumoniae
FtsW has been recently described [13]. Here we present
the results obtained by using ftsW^phoA and ftsW^bla
gene fusions in E. coli FtsW.
2. Materials and methods
2.1. Bacterial strains and growth conditions
The bacterial strains used were E. coli CC118 (araD139,
v(ara, leu)7697, vlacX74, vphoA20, galE, galK, thi, rpsE,
rpoB, argE (am), recA1), CC202 (F42, lacI3, zzf2: :
TnphoA/CC 118) and TG1 (FP traD36 lacIq v(lacZ)M15
pro Aþ Bþ /supE v(hsdM-mcrB)5 (rk 3mk 3mcrB3 ) thi
v(lac-proAB) that were grown in the Luria broth (LB)
medium at 37‡C. Antibiotics were used as follows : ampicillin, 100 Wg ml31 , and kanamycin, 30 Wg ml31 in liquid
media and 300 Wg ml31 in solid media.
5-Bromo-4-chloro-3-indolylphosphate disodium salt
(XP) (Sigma) was added at a concentration of 40 Wg
ml31 . p-Nitrophenyl phosphate (Sigma 104), a substrate
for alkaline phosphatase assay, also was from Sigma.
The ECF substrate (Amersham0 ) from the ECF (enhanced chemi£uorescence) signal ampli¢cation module
was purchased from Amersham and used at a concentration of 12 Wg ml31 for detection of the phosphatase activity of the fusions in intact cells.
2.2. Isolation of in vivo ftsW^phoA fusions
The strain CC202 was transformed with pBL1, a
pUC19 derivative plasmid containing a fragment of the
murD gene and the entire ftsW gene. As described by
Manoil and Beckwith [15], transformation was plated on
a medium containing ampicillin (100 Wg ml31 ), XP (40 Wg
ml31 ), and a high concentration of kanamycin (300 Wg
ml31 ) to isolate the colonies, in which transposition of
TnphoA into the multicopy plasmid occurred. After incubation at 37‡C, blue colonies were selected, and plasmid
DNA was puri¢ed and used to transform the strain
CC118. Selection was done on the same plates, and plasmid DNA was prepared and characterized by restriction
analysis and sequencing by standard methods [14].
FEMSLE 10621 31-10-02
2.3. Construction of gene fusions by ExoIII-nested deletions
Plasmid pJS521 (a gift of G.R. Jacobson, [16]) was ¢rst
digested with PstI and BamHI to clone the phoA gene in
pUC19 (we called this plasmid pBL8). pBL8 was treated
with HindIII, the 3P-recessed ends were ¢lled in by using
Klenow, and the subsequent digestion with SacI removed
the phoA-containing fragment. This fragment was then
ligated with pBL1 that was cut at the 3P end of the ftsW
gene (codon 402) with MluI, ¢lled in, and then digested
with SacI. The new plasmid was called pBL10 and was
used to construct a series of fusion plasmids by nested
deletions from the 3P end of the ftsW gene. Exonuclease
III (ExoIII) digests linear DNA with 5P overhanging ends
or blunt ends, whereas 3P overhanging ends are resistant to
the nuclease action. Then 7 Wg of pBL10 was cut with SalI
and placed within codon 388 to create a 5P overhanging
end that could be used as a substrate for the ExoIII;
digestion with PstI that creates a 3P overhang at the codon
27 (proline) of phoA was used to protect bases from
ExoIII. The digested DNA resuspended in 40 Wl of ExoIII
bu¡er was then incubated with 1 Wl of ExoIII at 37‡C.
Aliquots of 2.5 Wl were taken every 15 s, and placed on
ice in tubes containing 2.25 units of S1 nuclease. When all
aliquots had been taken, tubes were incubated for 30 min
at 30‡C. Then 1 Wl of S1 stop mixture (0.3 M Tris^HCl, 50
mM EDTA, pH 8) was added, and the tubes were heated
for 10 min at 70‡C. Klenow (0.125 units per tube) was
used to ¢ll in ExoIII-digested DNA in the presence of
deoxynucleotides (0.05 mM), and blunt ends were ligated
overnight at room temperature. DNA was transformed
into TG1 cells, and transformants were selected on LB
plates containing ampicillin (100 Wg ml31 ) and XP (40
Wg ml31 ). A restriction analysis was ¢rst made, and in
frame fusions were con¢rmed by sequencing.
2.4. In vitro construction of ftsW^phoA and ftsW^blaM
fusions
To complete FtsW topology, we made four in vitro
fusions at di¡erent amino acid positions of interest, which
de¢ne the topology more precisely. Fusion A82 was made
by digesting pBL10 with NruI (codon 82) and PstI, each
with a unique restriction site in the plasmid. Protruding
ends were made blunt by the Klenow enzyme, were ligated
together, and DNA was transformed ¢rst into TG1 cells
and then into CC118.
Fusions T38, P375, and L403 were constructed by replacing the ftsW gene in pBL10 with a polymerase chain
reaction (PCR) fragment coding the desired region to be
involved in the fusion. Three fragments, 600, 1590, and
1680 bp long, were ¢rst ampli¢ed, using oligonucleotides
A51 (5P-ACGCCAAGCTTGCATGCCGGG-3P) and A52
(5P-CATGATCAGGCTGCAGGTATCTTTTTCCCG-3P),
A54 (5P-ACCGTAACTGATCTGCAGCAATGTCAGACC-3P), A53 (5P-GCCTGCGCTTTCTGCAGACGCGTT-
Cyaan Magenta Geel Zwart
B. Lara, J.A. Ayala / FEMS Microbiology Letters 216 (2002) 23^32
TCA-3P), respectively, that contain a HindIII and a PstI
site, (underlined). This PCR fragments was cloned after
restriction in pBL10 also cut with PstI and HindIII, creating an in frame fusion with phoA at codons 40 (fusion
T38), 375 (fusion P375), and 403 (fusion L403) of the
ftsW gene.
To generate L-lactamase fusions we followed the method described by [17]. Various truncated forms of the ftsW
gene (at the 3P end) were ampli¢ed by PCR from the E. coli
chromosomal DNA using as primers oligonucleotides generating a KpnI site. The oligonucleotide A60 (5P-CGTACCAGGTACCGCCTGTGCCAGCAGTAACACTGC-3P)
used as the primer for the 5P end of the gene in all constructions introduced a BamHI site upstream from the
initiation codon of ftsW, within the sequence of the preceding murD gene [18]. The generated DNA fragments
were puri¢ed, cut with both BamHI and KpnI enzymes
and then inserted between the same sites of the pNF150
vector. In each case, the in frame insertion resulted in the
expression of a truncated form of the ftsW gene product
fused to the mature form of L-lactamase (HPETLVKVK_)
via a short intermediate peptide linker AVPHAISSSPLR
originating from the sequence of the pNF150 plasmid vector. In all cases, the site of fusion junction was con¢rmed
by DNA sequencing.
2.5. DNA sequence analysis of fusions
Double-stranded ftsW^phoA or ftsW^blaM fusion plasmid DNA was sequenced according to the Sequenase version 2.0 sequencing protocol (United States Biochemicals)
by the chain-terminator method [19]. The sequencing
primer hybridizing at the 5P end of PphoA (5P-CCCCCATCCCATCGCCAATCA-3P) was obtained from Isogen.
Deduced amino acid sequences were analyzed by using
the WISCONSIN computer programs and molecular biology services accessible on the World Wide Web: ExPASy
(http://expasy.hcuge.ch).
2.6. Alkaline-phosphatase activity of fusions
Activity of in frame fusions was measured as described
by Beckwith [20]. Overnight cultures were diluted 100-fold
in the LB medium and the appropriate antibiotic and allowed to grow at 37‡C to OD550 = 0.5. The cells were harvested and resuspended in the same volume of 1 M Tris^
HCl, pH 8. Phosphatase activity was assayed by measuring the rate of p-nitrophenyl phosphate hydrolysis.
The speci¢c activity of the fusion proteins was determined as the ratio of the measured phosphatase activity
to the amount of each fusion protein detected, at the exponential state of growth, as described below.
We developed a new assay to visualize the phosphatase
activity in intact cell, using a £uorescent substrate of
the Amersham ECF signal ampli¢cation module. This
substrate is a phospho-derivative that, when cleaved by
FEMSLE 10621 31-10-02
25
phosphatase, allows detection of £uorescence in the cell
envelope by £uorescence microscopy as well as the quanti¢cation of the activity in a spectro£uorometer (AMINCO Bowman series 2 luminescence spectrometer, excitation at 420 nm, emission at 560 nm). One ml of the
appropriate culture (109 cells ml31 ) was centrifuged for
10 min at 4‡C in an Eppendorf microfuge, 3000 rpm.
Cell pellet was resuspended in sodium bicarbonate 0.1 M
bu¡er, pH 7.5, washed once by centrifugation under the
same conditions as above, and ¢nally resuspended in 1 ml
of the same bu¡er. The ECL substrate (Amersham0 ) was
added to a ¢nal concentration of 12 Wg ml31 , and incubated for 10 min at 4‡C. Then 10 Wl of the cell suspension
were placed on a slide of agarose for observation and
photography in a £uorescence microscope (Olympus System Microscope BX50-FLA and Olympus Photomicrography System PM10AD). The rest was centrifuged for 10
min at 4‡C at 15 000 rpm to separate bacteria. Supernatant was used directly to measure the £uorescence emission at 560 nm, with an excitation wavelength of 420 nm
and a detector high voltage of 535 V.
2.7. Quanti¢cation of the expression of FtsW^PhoA fusions
The level of expression of each individual fusion protein
was quanti¢ed by immunoblotting of total exponential cell
culture. The CC118 strains containing plasmids harboring
in frame fusions were grown until late mid-exponential
phase. Total proteins were separated in the 10% acrylamide SDS^PAGE gel and transferred by blotting to Immobilon paper. The PhoA fragment in the fusion protein was
detected by hybridization of the membrane with a polyclonal anti-alkaline phosphatase antiserum and revealed
by the chemiluminescence method. Bands corresponding
to the fusion proteins were quanti¢ed (arbitrary units)
by densitometric analysis of the ¢lm, using a Mustek
1200SP scanner system and the software imaging-analysis
TINA version 2.09e.
2.8. Ampicillin resistance of cells expressing L-lactamase
fusion proteins
The ampicillin resistance of individual cells of DH5K
carrying the various ftsW^blaM plasmids listed in Table
1 was determined by plating appropriate dilutions of exponential phase cultures onto 2YT plates containing 0, 30,
60, 100 or 200 Wg ml31 ampicillin. Growth was observed
after 24 h of incubation at 37‡C, and the resistance was
estimated as the ratio of colony numbers on ampicillin
plates to colony numbers on control plates.
2.9. Transmembrane segment prediction
The following available programs on the World Wide
Web were used to predict the membrane topology of
FtsW: Tmpred, TMAP, SOSUI, DAS and pHDtopology
Cyaan Magenta Geel Zwart
26
B. Lara, J.A. Ayala / FEMS Microbiology Letters 216 (2002) 23^32
Table 1
Speci¢c activity of the FtsW^PhoA fusions obtained by two di¡erent methods
Fusion
T38
R73
A82
L100
A112
S118
L121
L126
S130
I146
V165
R172
V202
T207
V233
G274
Q279
Y290
E293
E306
L307
V322
F324
A326
Q355
P375
M387
L403
Method of
construction
PCR
ExoIII
Restr.
TnphoA
ExoIII
ExoIII
TnphoA
ExoIII
ExoIII
ExoIII
TnphoA
ExoIII
ExoIII
ExoIII
TnphoA
ExoIII
ExoIII
ExoIII
ExoIII
ExoIII
ExoIII
ExoIII
ExoIII
ExoIII
ExoIII
PCR
ExoIII
PCR
Junctiona
T38T-CS-PVL
R73R-PVL
A82G-PVL
L100P-[*]-PVL
A112G-PVL
S118C-PVL
L121P-[*]-PVL
L126R-PVL
S130S-PVL
I146S-PVL
V165A-[*]-PVL
R172R-PVL
V202G-PVL
T207S-PVL
V233A-[*]-PVL
G274G-PVL
Q279R-PVL
Y290C-PVL
E293G-PVL
E306G-PVL
L307R-PVL
V322G-PVL
F324C-PVL
A326G-PVL
Q355R-PVL
P375R-PVL
M387S-PVL
L403R-PVL
AP activity
units/ODb
15
279
119
5
0
3
3
73
133
226
3
60
118
97
25
0
1
1
10
1
0
36
30
29
4
131
109
9
Amount detected
with anti-PhoAc
9.3
41.6
25.2
2.7
6 0.1
1.6
6 0.1
13.6
13.7
26.7
3.22
5.7
29.1
22.1
2.8
0.4
8.7
33.4
10.2
19.2
8.2
3.42
16.6
48.7
0.5
14.3
39.1
12.0
Speci¢c AP
activity
1.61
6.70
4.72
1.85
0
1.87
3
5.37
9.70
8.46
0.93
10.5
4.05
4.39
8.93
0
0.11
0.03
0.98
0.05
0
10.5
1.81
0.60
8.00
9.16
2.78
0.75
Fluorescence
cellsd
supernatante
3
+++
+++
3
+/3
3
3
+++
+++
+++
3
++
+++
+
++
3
3
3
3
3
3
+
+
+/3
3
+++
+
3
4.25
37.07
39.60
13.5
10.5
7.0
15.0
34.57
36.01
35.65
2.50
36.11
38.18
29.81
38.05
0.70
2.28
5.34
2.62
3.45
4.06
25.06
14.43
5.57
0.21
37.71
30.84
5.61
a
Codon of ftsW into which PphoA is fused and the amino acid change produced, PVL indicates three ¢rst amino acids of PhoA, and [*] (DSYTQVASWTEPFPFC) is the amino acid sequence of the stretch introduced by the TnphoA derived from the IS50L insertion sequence.
b
The AP activity is the mean of two to ¢ve values from independent determinations.
c
The arbitrary unit obtained by densitometric analysis of the ¢lm, as described in Section 2.
d
Intensity of the yellow color of the cells after 10 min at 4‡C in the presence of the ECL substrate, as described in Section 2; 3 colorless, +/3 very
pale, + pale, ++ intense, +++ very intense.
e
Arbitrary units of the £uorescence intensity of the supernatants measured at 560 nm.
accessible on the World Wide Web: ExPASy (http://expasy.hcuge.ch). We used 58 homologous proteins to FtsW
identi¢ed in COG0772 database at the National Center for
Biological Information (http://www.ncbi.nlm.nih.gov/cgibin/COG).
3. Results and discussion
3.1. Prediction of the membrane topology of E. coli FtsW
The hydrophatic pro¢le of E. coli FtsW, previously
identi¢ed as a membrane protein, revealed three large hydrophobic regions (M1-V132, P181-A239, and L307-R414)
separated by two hydrophilic regions of 48 (K133-K180)
and 67 (E240-E306) residues, respectively. We could predict that the ¢rst N-terminal hydrophobic region contained three or four membrane-spanning segments, the
central hydrophobic region might contain from one to
three; and the third C-terminal hydrophobic region con-
FEMSLE 10621 31-10-02
tained three such segments. The two remaining parts of
the protein show a high degree of hydrophilicity and
should represent water-soluble domains (Fig. 1A).
The prediction of the secondary structure of the polypeptide chain by the Chou and Fasman [21] algorithm
con¢rmed that the most hydrophobic regions had a highly
K-helical nature. We also used ¢ve computer programs
available on the Word Wide Web (see Section 2) to predict
transmembrane segments. All ¢ve programs gave similar,
but not identical, predictions, ranging from nine to 11
transmembrane segments (TM) for E. coli FtsW, which
are summarized in Fig. 1A. A preliminary topological
model (called 10-TMS model) suggested by most of the
prediction methods contains 10 transmembrane segments,
0 (L13-S33), 1 (L48-M68), 2 (V87-W107), 3 (A112-V132),
5 (P181-A194), 6 (L198-G216), 7 (W220-A239), 8 (L307F324), 9 (G342-A364), 10 (T373-L395), two large hydrophilic loops, 3/5 (K133-K180), 7/8 (E240-E306) at the cytoplasm and periplasm, respectively, and both the N-terminus and C-terminus facing the cytoplasm (Fig. 1B).
Cyaan Magenta Geel Zwart
B. Lara, J.A. Ayala / FEMS Microbiology Letters 216 (2002) 23^32
27
Fig. 1. Predicted transmembrane segments of FtsW by di¡erent methods and the predicted and proposed models. A: Open bars represent transmembrane or uncharged segments. The prediction methods DAS, SOSUI, TMpred, TMAP, and pHDtopology are described in the text. The HYDROPHOBIC prediction was obtained by visual inspection of the sequence and represents segments of at least 14 uncharged residues, £anked by charged amino
acids. Ratios of positive to negative charged residues on the hydrophilic segments of the HYDROPHOBIC prediction (upper line) and pHDtopology
prediction (lower line) are shown. Marks + and 3 indicate the type of residue bordering the hydrophobic segments (+, R or K; 3, D or E). B: Predicted topological model of E. coli FtsW. C: Proposed model of E. coli FtsW. Numbers on the transmembrane segments correspond to those displayed
in panel A.
To check validity of this model, we used alkaline phosphatase and L-lactamase as topological reporters for analysis of topology of FtsW in the membrane.
3.2. Comparison with other putative FtsW proteins
We ¢rst performed the search on the SWISS-PROT,
release 40.12, TrEMBL, release 19.10 databases with the
BLAST program, using the sequence from E. coli ftsW
gene. One hundred and six homologous genes corresponding to putative FtsW or RodA proteins were identi¢ed by
this method, and also several genes by searching on the
Web page several other un¢nished sequence genome projects (http://www.ncbi.nlm.nih.gov:80/PMGifs/Genomes/
eub_u.html). Also, by HMM search at the TIGR databases (http://www.tigr.org), 83 homologs to E. coli FtsW
corresponding to 42 species and 32 major phylogenetic
lineages were identi¢ed, and the COG0772 database at
the National Center for Biological Information (http://
www.ncbi.nlm.nih.gov/cgi-bin/COG) contains 58 homolo-
FEMSLE 10621 31-10-02
gous proteins corresponding to 30 di¡erent species and 18
major phylogenetic lineages. Comparison analysis of the
amino acid sequence with the PILE-UP and DIVERGE
programs clearly separated those with the highest homology to FtsW protein from those with the strongest homology with RodA and divided the phylogenetic tree into two
sections (data not shown). This separation on the phylogenetic tree of the two families of homologous proteins
clearly indicates that, although the two proteins (FtsW
and RodA) must have quite similar functions, they have
remained as individual entities in the course of evolution.
This analysis also indicated that the gene products with
the strongest homology with E. coli FtsW are currently
located in a cluster of conserved cell division genes (dcw
cluster), in which there was also ftsZ or mraW. In those
species where a single FtsW/RodA homolog is found
(Buchnera sp. APS TOKIO 1998, Cyanophora paradoxa
(Cyanella), Deinococcus radiodurans R, Neisseria gonorrhoeae FA1090, Neisseria meningitidis Z2491, N. meningitidis MC58 and Synechocystis sp. PCC6803) or where
Cyaan Magenta Geel Zwart
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B. Lara, J.A. Ayala / FEMS Microbiology Letters 216 (2002) 23^32
there was a third homolog (Bacillus halodurans C-125,
Bacillus subtilis 168, Enterococcus faecalis V583, Lactococcus lactis IL1403, and Streptomyces coelicolor A3(2)) all
those proteins had a higher homology to FtsW than to
RodA.
Then we performed a comparison analysis among the
proteins, using the CLUSTAL-W program [22]. Although
the primary structures of the transmembrane segments
greatly diverge among orthologues (with the exception of
Ec-TM10), most of the predicted transmembrane segments
and loops are well conserved, in terms of hydrophobicity,
charge, number, and size, among the 29 orthologues
FtsW-like proteins (data not shown). This suggests that
general topology of all these proteins should be identical.
3.3. Activities of FtsW^PhoA and FtsW^BlaM fusion
proteins
In this work, we have applied a gene-fusion method [23]
to extensively investigate the membrane topology of the
essential E. coli cell division protein FtsW. The fusion
proteins and their activities are listed in Tables 1 and 2.
When activities of these fusion proteins were superimposed on the predicted 10-TMS model of FtsW, the results
obtained turned out to be incompatible with the predicted
topology (Fig. 1B). The reversed orientation of the EcTM1, Ec-TM2 and Ec-TM3 segments, the predicted
orientation for Ec-TM9 and Ec-TM10, and two large periplasmic and cytoplasmic loops were more strongly con¢rmed by the phoA fusion results. To further characterize
expression and localization of the PhoA moiety of the
fusion proteins, we developed a method to visualize the
alkaline phosphatase (AP) activity by £uorescence microscopy and to measure the activity by a £uorescence emis-
sion of a cleaved substrate (ECF £uorescence substrate of
the Amersham’s ECF signal ampli¢cation module, see
Section 2). After cleavage, the substrate can di¡use to
the periplasm and immediately outside the cell, and only
after 90^100 min it could also be found in the cytoplasm.
All those fusions with a £uorescence emission of at least
10.00 (arbitrary units) in the supernatant developed a £uorescence pattern on the cell envelope. Fig. 3 shows the
current pattern of £uorescence observed with FtsW^
PhoA fusion protein I146.
A high AP activity of fusion R73 (279 units/OD) and
A82 (119 units/OD) clearly indicates that loop 1/2 (13
residues) is located in the periplasm, while the low activity
of fusion T38 (15 units/OD) suggests that loop 0/1 (13
residues) must be in the cytoplasm. According to the positive-inside von Heijne’s rule [24], topology of these loops
is in a good agreement with the ratio of positive to negative charge of 3:2 and 8:4, respectively. These data con¢rm the idea that Ec-TM1, and not Ec-TM0, could be the
¢rst topological signal for insertion of FtsW and the ¢rst
anchoring segment to the membrane. The homology comparison analysis of the N-terminal region of E. coli FtsW
(M1-R46) with 29 FtsW-homologous proteins reveals no
homologous hydrophobic sequence to the ¢rst predicted
transmembrane segment (Ec-TM0, L13-S33), so we postulate that this region should remain in the cytoplasm.
Moreover, a FtsW form of a lower molecular mass
(FtsWS) has a start of translation (M42) after this segment, and this form seems to be completely functional in
vivo [11], which indicates no essential role of this region,
nor topogenic determinant for Ec-TM0. However, we cannot exclude that this region, in the case of E. coli FtsW,
could be associated with the membrane after insertion of
the protein.
Table 2
Construction of the ftsW^blaM gene fusions and response of fusion-containing cells to ampicillin
Plasmid
pFTSWB1
pFTSWB2
pFTSWB3
pFTSWB4
pFTSWB5
pFTSWB6
pFTSWB7
pFTSWB8
pFTSWB9
Oligonucleotidea
3P end PCR primer
5P-CGGTGGTACCGCGCCGCGCAGGTTATTACG-3P
5P-CGTACCAGGTACCGCCTGTGCCAGCAGTAACACTGC-3P
5P-CGTAGGTACCGCTGGCTGTGCCAGCAGTAACACTGC-3P
5P-CTGCGGTACCGCCGCTCCCGCCAGGAACAAC-3P
5P-GGCAATGGGTACCGCCAATTTCGCTCCCGCCAG-3P
5P-CGGCGGGGTACCGCCGGTTCGGCGAGTATCAG-3P
5P-CTGAGGTACCGCGCCAAAGGGATCTTCCCACGG-3P
5P-ACGGGGTACCGCCATCGCGCGAAAAGCGAC-3P
5P-GGTCAGGTACCGCTGCTTTACGGCCAATCG-3P
5P end PCR primer
5P-AGCCTGGATCCGTTCAAGAACTTTGAACAACG-3P
Junction siteb
Response to ampicillinc
G-177
Q-195
P-196
A-217
L-219
P-241
G-259
M-327
A-333
S
R
R
S
S
S
S
S
S
a
Restriction sites for BamHI (GGATCC) and KpnI (GGTACC) that were introduced in oligonucleotide sequences are indicated in bold.
The junction site corresponds to the C-terminal amino acid of the truncated FtsW sequence, which has been fused to the mature form of Llactamase,
via an intermediate peptide linker consisting of AVPHAISSSPLR (originating from the pNF150 vector sequence).
c
The DH5K E. coli strains harboring plasmids bearing fusions between complete or truncated ftsW from E. coli and blaM were grown as 100^200 separate single colonies on plates containing ampicillin at 100 Wg ml31 and kanamycin at 50 Wg ml31 . After 24 h of incubation at 37‡C, strains growing in
the presence of 100 Wg ml31 ampicillin were considered as resistant (R) and otherwise sensitive (S). Strains harboring plasmids pFTSWB2 and
pFTSWB3 also grew at 200 Wg ml31 . Strains harboring plasmids pFTSWB1, pFTSWB4, pFTSWB5, pFTSWB6, pFTSWB7, pFTSWB8 and pFTSWB9
did not grow at 30 Wg ml31 .
b
FEMSLE 10621 31-10-02
Cyaan Magenta Geel Zwart
B. Lara, J.A. Ayala / FEMS Microbiology Letters 216 (2002) 23^32
29
Fig. 2. Sequence alignment of FtsW from S. pneumoniae and E. coli. The alignment was obtained by the CLUSTAL-W program. Identical or homolog
amino acids are marked with a vertical bar and two dots, respectively. Predicted transmembrane segments for S. pneumoniae (Sp-TM) and E. coli (EcTM) are marked as black bars under each sequence. Open (inactive) or black (active) triangle shows the positions of PhoA fusion. The positions of Bla
fusion are shown as open (sensitive) or black (resistant) diamond. For comparison, the positions of the PhoA fusions recently described in S. pneumoniae FtsW are also shown.
The overall topology and midpoint of the actual transmembrane region Ec-TM3 in Fig. 4 was inferred from the
activity results, such as an increasing AP activity from the
N- to the C-terminal of the segment in fusions A112, (0
unit/OD); S118, (3 units/OD); L121, (3 units/OD); L126,
(73 units/OD) ; and S130, (133 units/OD). This is also
con¢rmed by the published evidence that as few as about
10 residues of an ‘outgoing’ transmembrane segment are
su⁄cient to promote export when fused to AP, whereas 10
residues of an ‘in-coming’ transmembrane region usually
are insu⁄cient to translocate AP into the cytoplasm [25].
This e¡ect of the insertions on the potential location of the
catalytic activity can be observed in the case of fusions
A112, S118, and L121 that clearly disrupt the transmembrane segment, while fusions L126 and S130 allow export
of the PhoA moiety. If so, the loop 2/3 (¢ve residues) and
loop 3/4 (11 residues) should be located in the cytoplasm
and in the periplasm, respectively.
FEMSLE 10621 31-10-02
The central region of the protein contains three potential transmembrane segments. However, if we consider
that the loops should have at least ¢ve residues, the predicted loop 5/6 is too small, and also the length of EcTM5 is rather short. The high AP activities of fusion proteins from S130 (speci¢c AP activity 9.70) to T207 (speci¢c
AP activity 4.39) allowed us to think that this entire segment should be in the periplasm. However, the resistance
pattern of strains harboring plasmids pFTSWB1 (sensitive), pFTSWB2 (resistant) and pFTSWB3 (resistant), permits us to consider the Ec-TM5 as an actual transmembrane segment with the predicted orientation (see Fig. 1).
Comparison with the corresponding transmembrane segment (Sp-TM5) in S. pneumoniae (Fig. 2) con¢rms this
assumption and allows to extend the length of the segment
up to G177. In this context, a new transmembrane segment Ec-TM4, only predicted by pHDtopology, has been
identi¢ed by AP fusions I146 (AP activity 226) and V165
Cyaan Magenta Geel Zwart
30
B. Lara, J.A. Ayala / FEMS Microbiology Letters 216 (2002) 23^32
(AP activity 3). Considering those AP activities, this segment should have the ‘out-in’ orientation and it must correspond to Sp-TM4 displayed in Fig. 2. High AP activity
of fusion R172 (AP activity 60) can be explained by the
high charged segment preceding the fusion point that
should prevent Ec-TM4, already a charged segment, to
function as in-going transmembrane segment.
The predicted topology for Ec-TM6 and Ec-TM7 could
be con¢rmed by the activity and resistant pattern of AP
fusions V202 (AP activity 118), T207 (AP activity 97),
V233 (AP activity 25) and BlaM fusions on plasmid
pFTSWB4 (A217) (sensitive) and pFTSWB5 (L219) (sensitive). These results corroborate the actual orientation
‘out-in’ of Ec-TM6 and ‘in-out’ of Ec-TM7 and are in
agreement with the corresponding transmembrane segment of S. pneumoniae, Sp-TM6 and Sp-TM7, respectively.
At the C-terminal of the protein there is a region from
E240 up to F340, where all the fusions, both PhoA and
BlaM, give low AP activity or sensitive colonies. These
results could be interpreted as that the whole region is
located in the cytoplasm, but this topology is at odds
with the rest of model. The major assumption in the fusion
approach is that truncation of the membrane protein does
not a¡ect its native topology. However in our analysis, as
it has been already shown in the case of S. pnemoniae
FtsW [13], the PhoA and BlaM fusions failed to con¢rm
the predicted topology of this C-terminal region. This remark is consistent with the possibility that the correct
insertion of the loop 7/8 and transmembrane Ec-TM8 is
dependent on or requires interactions of protein segments
located downstream of the fusion sites. In this sense there
Fig. 3. Detection of the AP activity in the cell envelope by £uorescence
microscopy. One ml of an LB culture (109 cells ml31 ) of the strain
CC118/pBL10(I146) was centrifuged in an Eppendorf microfuge. Cell
pellet was washed and resuspended in the 0.1 M sodium bicarbonate
bu¡er, pH 7.5, and the ECL substrate (Amersham0 ) was added and incubated for 10 min at 4‡C. Then 10 Wl of the cell suspension were
placed on a slide of agarose for observation by phase contract (A) and
£uorescence (B) microscopy. The bar in panel A represents 2 Wm.
FEMSLE 10621 31-10-02
are two cysteine residues, one located at the cytoplasmic
inlet within Ec-TM4 (C158) and the other in Ec-TM9
(C346), that could establish a covalent link between these
two transmembrane segments and be required for stabilization. Deletion of these sequences may prevent or decrease the probability of the correct insertion. Indeed,
PhoA fusions in the most C-terminal part of FtsW, from
Q355 to the C-terminus, may be adopting the correct topology. Thus, the periplasmic location of loop 7/8 and the
right prediction for Ec-TM8 have been inferred from the
high homology of this region (50% identity, 71% homology) with the corresponding segment (loop 7/8 and SpTM8) of the published topology of S. pneumoniae FtsW
[13].
In the last segment of the protein (F340-R414) the high
AP speci¢c activity of fusion P375 (9.16) allows us to
consider the right prediction of the loop 9/10 in the periplasm, whereas the low activity of fusion L403 (0.75) predicts cytoplasmic location of the C-terminal end of the
protein. Location of the C-terminal end of the protein in
the cytoplasm also conforms to the positive-inside von
Heijne’s rule (ratio 5:3) [24]. Considering the cytoplasmic
location of loop 8/9 and the C-terminal end, and the periplasmic location of loop 9/10, the predicted ‘in-out’ orientation of transmembrane Ec-TM9 and the ‘out-in’ orientation of transmembrane Ec-TM10 are expected. This
topology for Ec-TM9 and Ec-TM10 is also con¢rmed by
a low £uorescence of fusion Q355 (0.21) and a relatively
high £uorescence of fusion M387 (30.84). A high activity
of the last fusion M387 indicates disruption of the ‘ingoing’ K-helix of Ec-TM10, which allows the PhoA moiety
to remain in the periplasm.
When all the fusions were analyzed by the in vivo £uorescence method, the data correlated precisely (see Table
1) with those obtained by the colorimetric method. However, a lower activity for fusion Q355 (0.21) was found,
which may result from a disruption of the out-going Khelix Ec-TM9 retaining PhoA in the cytoplasm. A higher
AP speci¢c activity detected for this fusion by the colorimetric method was actually due to the low level of the
fusion detected by immunoblotting of the total cell extract,
as the AP activity also was low.
From these data, we propose a topological model with
10 transmembrane segments and a large periplasmic loop
(Fig. 4). The topological model would have the following
structure: transmembrane segments (Ec-TM1 : T47-A66,
Ec-TM2 : G86-M104, Ec-TM3: Y110-G129, Ec-TM4 :
L141-N162, Ec-TM5 : G177-Q195, Ec-TM6 : T200-G216,
Ec-TM7 : W220-A239, Ec-TM8 : L307-F324, Ec-TM9 :
F343-A364, and Ec-TM10: L374-L394), cytoplasmic segments (N-terminal: M1-R46, loop 2/3: E105-R109, loop 4/
5: Y163-R176, loop 6/7: A217-L219, loop 8/9: R325G342, C-terminal: L395-R414) and periplasmic loops
(loop 1/2: S67-D85, loop 3/4: S130-D140, loop 5/6:
P196-G199, loop 7/8: E240-E306, loop 9/10: G365T373]. Although this proposed model (Figs. 1C and 4) is
Cyaan Magenta Geel Zwart
B. Lara, J.A. Ayala / FEMS Microbiology Letters 216 (2002) 23^32
31
Fig. 4. Membrane topology model of FtsW from E. coli. Predicted topological model derived from AP activity and L-lactam resistance of the fusion
proteins and by comparison with the predicted model of FtsW of S. pneumonia. The primary amino acid sequence is given in the single-letter code and
in black characters surrounded by a circle, a square or a diamond. Residues in squares indicate a conserved position in at least 60% of the FtsW homologs, while those in diamonds are conserved in more than 90% of the FtsW homologs proteins. Plus and minus signs indicate positively and negatively
charged amino acids, respectively. Amino acids corresponding to predicted transmembrane segments are in gray. The positions (in white characters) of
the PhoA and BlaM fusions are superimposed to the model. Fusions are indicated according to the AP activity or Bla resistance as black (active or resistant) or gray (inactive or sensitive) background. Positions of the known amino acid change mutations are also indicated : E170K, P181L, and P253L.
Conserved residues among the sequence of FtsW from E. coli and S. pneumonia are highlighted by a bold line and white background.
at odds with the results of hydrophobicity analysis, which
predicts some transmembrane segments not present in our
¢nal model (Ec-TM0) and the reversed orientation of
some others (Ec-TM1, Ec-TM2 and Ec-TM3), the consistency of the properties of the fusions (steady-state levels,
PhoA activities and BlaM resistance, and £uorescence on
the cell membrane), as well as the high correlation of our
results with those previously published for S. pnemoniae
FtsW (see Fig. 2), allow us to be con¢dent about the
model. These data suggest a general similar topology for
all member of the FtsW/RodA/SpoVE family.
Acknowledgements
This work was supported by grants BIO97-0665 and
PB97-1193 from the Direccio¤n General de Ensen‹anza
Superior e Investigacio¤n Cient|¤¢ca (Ministerio de Educacio¤n y Cultura, Spain). The ¢nancial support of Fundacio¤n Ramo¤n Areces to the Centro de Biolog|¤a Molecular
‘Severo Ochoa’ is greatly acknowledged.
FEMSLE 10621 31-10-02
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