Role of leucine zipper motifs in association of the Escherichia coli cell division proteins FtsL and FtsB.
ABSTRACT FtsL and FtsB are two inner-membrane proteins that are essential constituents of the cell division apparatus of Escherichia coli. In this study, we demonstrate that the leucine zipper-like (LZ) motifs, located in the periplasmic domain of FtsL and FtsB, are required for an optimal interaction between these two essential proteins.
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ABSTRACT: FtsB and FtsL are two essential integral membrane proteins of the bacterial division complex or "divisome", both characterized by a single transmembrane helix and a juxta-membrane coiled coil domain. The two domains are important for the association of FtsB and FtsL, a key event for their recruitment to the divisome, that in turn enables recruitment of the late divisomal components to the Z-ring and subsequent completion of the division process. Here we present a biophysical analysis performed in vitro that shows that the transmembrane domains of FtsB and FtsL associate strongly in isolation. Using FRET, we have measured the oligomerization of fluorophore-labeled transmembrane domains of FtsB and FtsL in both detergent and lipid. The data indicates that the transmembrane helices are likely a major contributor to the stability of the FtsB-FtsL complex. Our analyses show that FtsB and FtsL form a 1:1 higher-order oligomeric complex, possibly a tetramer. This finding suggests that the FtsB-FtsL complex is capable of multi-valent binding to FtsQ and other divisome components, a hypothesis that is consistent with the possibility that the FtsB-FtsL complex has a structural role in the stabilization of the Z-ring.Biochemistry 10/2013; · 3.38 Impact Factor
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ABSTRACT: The peptidoglycan cell wall of bacteria is a complex macromolecule composed of glycan strands that are crosslinked by short peptide bridges. Its biosynthesis involves a conserved group of enzymes, the bifunctional penicillin-binding proteins (bPBPs), which contain both a transglycosylase and a transpeptidase domain, thus being able to elongate the glycan strands and, at the same time, generate the peptide crosslinks. The stalked model bacterium Caulobacter crescentus possesses five bPBP paralogs, named Pbp1A, PbpC, PbpX, PbpY and PbpZ, whose function is still incompletely understood. In this study, we show that any of these proteins except for PbpZ is sufficient for growth and normal morphogenesis when expressed at native or elevated levels, whereas inactivation of all five paralogs is lethal. Growth analyses indicate a central role of PbpX in the resistance of C. crescentus against the non-canonical amino acid D-alanine. Moreover, we show that PbpX and PbpY localize to the cell division site. Their recruitment to the divisome is dependent on the essential cell division protein FtsN and likely involves interactions with FtsL and the putative peptidoglycan hydrolase DipM. The same interaction pattern is observed for Pbp1A and PbpC, although these proteins do not accumulate at midcell. Our findings demonstrate that the bPBPs of C. crescentus are, to a large extent, redundant and have retained the ability to interact with the peptidoglycan biosynthetic machineries responsible for cell elongation, cytokinesis and stalk growth. Nevertheless, they may preferentially act in specific peptidoglycan biosynthetic complexes, thereby facilitating the independent regulation of distinct growth processes.Journal of bacteriology 02/2014; · 3.94 Impact Factor
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ABSTRACT: Escherichia coli cell division is effected by a large assembly of proteins called the divisome, of which a subcomplex consisting of three bitopic inner membrane proteins, FtsQ, FtsB and FtsL, is an essential part. These three proteins, hypothesized to link cytoplasmic to periplasmic events during cell division, contain large periplasmic domains that are of major importance for function and complex formation. The essential nature of this subcomplex, its low abundance and its multiple interactions with key divisome components in the relatively accessible periplasm make it an attractive target for the development of protein-protein interaction inhibitors. Although the crystal structure of the periplasmic domain of FtsQ has been solved, the structure of the FtsQBL complex is unknown with only very crude indications of the interactions in this complex. In this study, we used in vivo site-specific photo cross-linking to probe the surface of the FtsQ periplasmic domain for its interaction interfaces with FtsB and FtsL. An interaction hotspot for FtsB was identified around residue S250 in the C-terminal region of FtsQ and a membrane proximal interaction region for both proteins around residue K59. Sequence alignment revealed a consensus motif overlapping with the C-terminal interaction hotspot underlining the importance of this region in FtsQ. The identification of contact sites in the FtsQBL complex will guide future development of interaction inhibitors that block cell division.Journal of Biological Chemistry 07/2013; · 4.65 Impact Factor
JOURNAL OF BACTERIOLOGY, Sept. 2011, p. 4988–4992
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 193, No. 18
Role of Leucine Zipper Motifs in Association of the Escherichia coli
Cell Division Proteins FtsL and FtsB?†
Carine Robichon,1‡ Gouzel Karimova,1Jon Beckwith,2and Daniel Ladant1*
Institut Pasteur, CNRS URA 2185, Unite ´ de Biochimie des Interactions Macromole ´culaires, De ´partement de
Biologie Structurale et Chimie, 28 rue du Dr Roux, 75724 Paris Cedex 15, France,1and
Department of Microbiology and Molecular Genetics, Harvard Medical School,
200 Longwood Avenue, Boston, Massachusetts 021152
Received 7 March 2011/Accepted 8 July 2011
FtsL and FtsB are two inner-membrane proteins that are essential constituents of the cell division apparatus
of Escherichia coli. In this study, we demonstrate that the leucine zipper-like (LZ) motifs, located in the
periplasmic domain of FtsL and FtsB, are required for an optimal interaction between these two essential
The FtsB, FtsL, and FtsQ proteins play a key role in the
assembly of the cell division machinery in both Gram-negative
and Gram-positive bacteria (1, 14, 30). They form a trimeric
complex in the inner membrane that is centrally located in the
sequential recruitment pathway of the divisomal proteins in
Escherichia coli (5, 15). This complex connects the upstream
cell division components, which are mainly cytosolic and as-
sembled around the FtsZ ring, with the downstream partners
that are mainly membrane-associated or periplasmic proteins
(8, 9, 14, 30). FtsL and FtsB can form a subcomplex indepen-
dently of FtsQ (6, 15, 18, 21) and are mutually dependent for
their stabilization: FtsL requires FtsB to be stable whereas
FtsB is partially degraded when FtsL is depleted (7, 18). No-
ticeably, a regulated proteolytic degradation of these proteins
might be involved in the control of cell division (4, 31). It was
previously shown that FtsL and FtsB interaction is mediated
mainly by their transmembrane domains and the membrane-
proximal portion of their periplasmic domains, while the C-
terminal portion of their periplasmic domains is involved in
their interaction with FtsQ (5, 16–18, 29). Both FtsL and FtsB
proteins contain, in their periplasmic part, a leucine zipper
(LZ) motif present in all orthologous proteins, although these
proteins are usually poorly conserved (7, 13, 17). The LZ motif
was originally suspected to mediate the FtsL-FtsB het-
erodimerization (5, 7, 28), although it was shown that in Ba-
cillus subtilis, FtsL variants modified in the heptad repeat were
functional (27). More recently, Gonzalez and Beckwith re-
ported that in E. coli, a truncated form of FtsB, lacking about
half of the LZ, was still able to associate with FtsL (18), thus
questioning the precise contribution of this motif in the inter-
action between these molecules. Here we reexamined this issue
by analyzing FtsL and FtsB variants modified in their LZ
motifs. The functionality of these variants was assessed by their
ability to complement the absence of wild-type copies of the
corresponding proteins. Their association capacities were
tested by bacterial two-hybrid (BACTH) and coimmunopre-
Modification of the leucine zipper motifs of FtsB and FtsL
affects their interaction. We generated FtsB and FtsL variants,
called FtsBMand FtsLM, in which the key leucines L46, L53,
L60, and L67 of FtsB and L63, L70, L77, and L84 of FtsL were
replaced with alanines (see the supplemental material). These
modifications should destabilize the hydrophobic interface
formed between the two LZ motifs without affecting the over-
all alpha-helical structure of the coiled coil, thus avoiding a
dramatic alteration of the proteins that could lead to their
instability and degradation (7, 18). The impact of these mod-
ifications was assessed by using the BACTH assay that has
been previously used to analyze interactions between Fts pro-
teins (2, 10, 11, 21, 23). In this assay, the proteins of interest are
fused to two complementary fragments, T25 and T18, from the
adenylate cyclase of Bordetella pertussis and expressed in an E.
coli cya strain. Upon interaction between the two proteins of
interest, the fused T25 and T18 fragments reconstitute a chi-
meric adenylate cyclase that produces cyclic AMP (cAMP),
which in turn activates ?-galactosidase (?-Gal) synthesis (22).
Genes encoding the two modified variants, FtsLMand FtsBM,
as well as the wild-type counterparts, were fused to the C
terminus of either T25 or T18 and expressed from the BACTH
vector pKT25 or pUT18C, respectively, under the control
of isopropyl-?-D-thiogalactopyranoside (IPTG)-inducible plac
promoters (see the supplemental material) (24).
To probe the functionality of the hybrid proteins, in vivo
complementation assays were carried out in an E. coli FtsL or
FtsB depletion strain (NB988 and CR14, respectively ), in
which the corresponding chromosomal gene is inactivated,
while a complementing ftsL or ftsB allele is expressed from a
pBAD plasmid (19) (see the supplemental material) by arabi-
nose induction. Figure 1 shows that expression of T18-FtsLM
or T18-FtsBMfusion restored the viability of the depleted
* Corresponding author. Mailing address: Institut Pasteur, CNRS
URA 2185, Unite ´ de Biochimie des Interactions Macromole ´culaires,
De ´partement de Biologie Structurale et Chimie, 28 rue du Dr Roux,
75724 Paris Cedex 15, France. Phone: 33145688400. Fax: 33140613042.
† Supplemental material for this article may be found at http://jb
‡ Present address: New England Biolabs, 240 County Road, Ipswich,
?Published ahead of print on 22 July 2011.
strain NB988 or CR14—in the absence of the wild-type FtsL or
FtsB—with the same efficiency as the wild-type T18-FtsL or
T18-FtsB fusion, indicating that they were all functional. Fur-
ther experiments using green fluorescent protein (GFP) fu-
sions showed that both variants, FtsLMand FtsBM, could be
recruited to the divisome (data not shown), suggesting that the
Leu-to-Ala changes in the LZ motif of FtsL or FtsB had no
drastic effect on the protein structures and functions, in agree-
ment with an earlier report (27).
The BATCH assay was then performed to characterize the
interaction properties of the different hybrids. The various T18
and T25 fusions were coexpressed in the E. coli cya strain
DHM1 (21), and the interaction efficiencies were quantified by
measuring ?-Gal activities. Associations of the different fu-
FIG. 1. Functional assays of the Leu-to-Ala variants of FtsB and FtsL. The modified genes ftsLMand ftsBMwere chemically synthesized by
Geneart AG (Regensburg, Germany) and subcloned into pKT25 and pUT18C as described in the supplemental material. (A) Complementation
assays of the T18 fusion proteins were performed in the FtsB- or FtsL-depleted strain (CR14 or NB988 , respectively) as described in the
supplemental material, by spotting 5 ?l of each overnight preculture diluted 10?1to 10?6on NZY solid medium containing IPTG (100 ?M) and
either arabinose (ARA) to induce expression of the pBAD-regulated gene or glucose (GLU) to repress its expression. Plates were then incubated
for 24 h at 37°C. (B and C) Western blotting assays were performed as previously described (26) on total extracts from cells grown in the presence
of arabinose or glucose after 1 h of induction of the T18 fusion construct with 100 ?M IPTG. The Western blots were probed with anti-FtsL or
anti-FtsB antibodies (26) or with an anti-T18 monoclonal antibody (see the supplemental material). The strains used are the FtsB-depleted strain
CR14 or the FtsL-depleted strain NB988 harboring pUT18C derivatives expressing the indicated hybrid proteins. Note that the T18 fusions are
significantly less abundant in the cells grown in the presence of glucose, probably because the plac promoter that drives the transcription of the
T18 hybrids is under catabolite repression (24).
VOL. 193, 2011NOTES 4989
sions with FtsQ were tested as a control (5, 18). As shown in
Fig. 2, the mutations within the LZ motif of both FtsL and
FtsB had no significant effect on their interactions with FtsQ,
indicating that the overall structure of FtsLMand FtsBMC-
terminal domains had not been altered by the Leu-to-Ala
replacements. However, the hybrid protein T25-FtsBMinter-
acted less efficiently with T18-FtsL than did the wild-type T25-
FtsB fusion, as suggested by the lower level of ?-Gal activity.
Similarly, the interaction efficiency of T25-FtsB with T18-FtsLM
was about 40% lower than that measured with T18-FtsL. Im-
portantly, cells coexpressing T18-FtsLMand T25-FtsBMexhib-
ited an even lower ?-Gal activity, corresponding to about 25%
of that measured in DHM1/T18-FtsL/T25-FtsB (Fig. 2). Thus,
the Leu-to-Ala replacement within the LZ motifs of both FtsL
and FtsB weakened their association, indicating that the hep-
tad leucine repeats in both proteins contribute to the stabili-
zation of the FtsL-FtsB heterodimer.
Western blot analysis of T18 (Fig. 2A) and T25 (Fig. 2B)
fusion stability revealed that the protein amounts were in good
correlation with the levels of measured ?-Gal activities. In-
deed, the hybrid proteins were unstable in the absence of an
interacting partner, consistent with the fact that FtsL and FtsB
costabilize each other (18), but were much more stable when
coexpressed with FtsQ.
The two-hybrid interaction data were further confirmed by
coimmunoprecipitation. For this, FtsL or FtsLMfused to GFP
(on the low-copy-number plasmid pNG162 ) was coex-
pressed in the FtsB depletion strain CR14 with FtsB or FtsBM
tagged with a Flag epitope (26). These variants (FtsBflag3or
FtsBMflag3) were expressed from a pDSW204 plasmid inte-
grated into the chromosome (3, 32). Immunoprecipitations
were carried out on bacterial membrane extracts using anti-
Flag M2 affinity beads, and immunoprecipitated complexes
were then probed by Western blotting with anti-GFP antibod-
ies. As shown in Fig. 2C, the GFP-FtsL fusion was efficiently
immunoprecipitated by both the FtsBflag3and FtsBMflag3poly-
peptides (Fig. 2C, lanes 5 and 7). In contrast, the GFP-FtsLM
fusion was immunoprecipitated with a lower efficiency by the
wild-type FtsBflag3and, more importantly, GFP-FtsLMwas not
immunoprecipitated by FtsBMflag3(Fig. 2C, lanes 6 and 8).
These results demonstrated that the Leu-to-Ala modifications
in the LZ of both FtsL and FtsB significantly decreased their
association, thus corroborating the conclusion of the two-hy-
Swapping of the leucine zipper domain of FtsL and FtsB
with the analogous domain from Haemophilus influenzae FtsL
and FtsB orthologs. To further delineate the polypeptide re-
gions that mediate the specificity of association between FtsL
and FtsB, we applied a “domain swapping” approach (12, 20).
For that, we selected the orthologous FtsL and FtsB proteins
from H. influenzae, FtsLhinf and FtsBhinf. FtsLhinf shares
about 30% amino acid sequence identity with E. coli FtsL,
while FtsBhinf is about 40% identical with E. coli FtsB (Fig.
3A). Ghigo and Beckwith (12) previously reported that FtsL-
hinf could not complement the E. coli FtsL null mutant and did
not localize to the septum. However, replacement of the E. coli
FtsL LZ domain with the corresponding sequence of FtsLhinf
resulted in a hybrid protein, LLhinfL, that was able to com-
plement the absence of FtsL, although less efficiently than the
native FtsL protein (12).
The FtsLhinf and FtsBhinf coding regions were PCR ampli-
fied and cloned into the pKT25 and pUT18C vectors (see the
supplemental material). We also analyzed the recombinant
protein LLhinfL, in which the LZ region of E. coli FtsL was
replaced with the corresponding sequence of FtsLhinf (12).
Similarly, a BBhinfB variant was constructed by replacing the
LZ region of FtsB with the corresponding region of H. influ-
enzae FtsBhinf (Fig. 3B). The resulting T18 hybrid proteins
were first characterized by functional complementation assays
in E. coli depletion strains lacking either FtsL or FtsB. We
found that T18-LLhinfL and, more surprisingly, T18-FtsLhinf
could partially restore the growth of the E. coli FtsL-depleted
strain NB988 (Fig. 1A), in variance from the previous results of
Ghigo and Beckwith (12). This may be due to a higher expres-
sion level of the T18-Lhinf protein achieved with the high-
FIG. 2. Interactions of the FtsB and FtsL LZ variants studied by
BACTH assay and coimmunoprecipitation. (A and B) ?-Galactosidase
activities in the E. coli DHM1 strain coexpressing T18 and T25 fusion
proteins were measured on liquid cultures as described in the supple-
mental material. Results are expressed as relative units with standard
deviations (SD) indicated in parentheses. Western blotting assays were
performed as described previously (23) on total cell extracts from the
same cultures used for the ?-galactosidase assays using anti-T18 or
anti-T25 antibodies (see the supplemental material) (B). The asterisk
indicates an unspecific band. (C) Coimmunoprecipitations (IP, lanes 3
to 8) were performed as described in the supplemental material with
anti-Flag M2 affinity beads (Sigma) on solubilized membrane protein
extracts from the E. coli FtsB-depleted strain CR14 (26) coexpressing
the indicated GFP fusions (GFP-FtsL or GFP-FtsLM, expressed from
plasmid pNG162) and Flag fusion proteins (FtsBflag3or FtsBMflag3,
expressed from plasmid pDSW204 integrated onto the chromosome at
the ?att site) (see the supplemental material). The Western blot was
probed with anti-GFP antibodies. Lane 1 is a total extract (TE) of
strain CR14 coexpressing GFP-FtsL and FtsBflag3. Lane 2 corresponds
to a control IP performed with strain CR14 coexpressing GFP-FtsL
and the Flag3 epitope only (i.e., not fused to FtsB). The asterisk
indicates an unspecific band.
copy-number vector pUT18C (pUC19 origin) while, in the
earlier study, FtsLhinf was expressed from a low-copy-number
plasmid (pSC101 origin) (12). Similarly, the T18-FtsBhinf fu-
sion and T18-BBhinfB swap construct were also able to com-
plement the lack of FtsB (Fig. 1A). Altogether, these data
indicated that, when produced from a high-copy-number vec-
tor, the wild-type FtsB and FtsL from H. influenzae as well as
the BBhinfB and LLhinfL variants were functional in E. coli.
Interactions between these hybrid proteins were then char-
acterized by BACTH assays. As shown in Fig. 3C, FtsLhinf and
FtsBhinf heterodimerized, although less efficiently than their
E. coli orthologs (?9 ?-Gal units versus ?22 units, respec-
tively). Interaction of FtsLhinf with E. coli FtsB was reduced
(?6 units), while E. coli FtsL did not associate with FtsBhinf
(?2 units), revealing an intraspecies selectivity of association
between these two components of the divisome. Replacing the
LZ motif of FtsL with the corresponding region of FtsLhinf
restored the association of the hybrid protein T18-LLhinfL
with T25-FtsBhinf to a level similar to that found for the
FtsLhinf/FtsBhinf heterodimerization (9.5 ?-Gal units versus 9
units, respectively). The converse replacement of the LZ of
FtsB with the corresponding region of FtsBhinf (T25-
BBhinfB) did not restore association with T18-FtsLhinf (2.3
units) but significantly enhanced the interaction with the
LLhinfL (T18-LLhinfL) variant compared to the wild-type E.
coli FtsL (T18-FtsL) (12.5 versus 6.7 units). This confirmed the
intraspecies selectivity of association of the LZ motifs of FtsL
In control experiments, the different hybrids were assayed
for interaction with E. coli FtsQ protein. FtsLhinf and LLhinfL
both associated efficiently with FtsQ, while FtsQ interaction
with FtsBhinf was reduced compared to E. coli FtsB or
BBhinfB. As the C-terminal end of FtsBhinf diverges largely
from that of E. coli FtsB (Fig. 3A), these results support the
idea that the C-terminal extremity of FtsB is involved in FtsQ
binding as recently proposed (18). Altogether, the present data
FIG. 3. BACTH analysis of FtsBhinf, FtsLhinf, and LZ swap variants. (A) The amino acid sequence alignment of FtsB and FtsBhinf is shown
with single-letter abbreviations. The predicted coiled-coil domain of FtsB and FtsBhinf is framed in gray, whereas the leucine zipper motifs are
boxed in gray. The proposed cytoplasmic (Cyto), transmembrane (TM), and periplasmic domains of each protein are underlined. An alignment
of FtsL and FtsLhinf is available in reference 12. (B) Schematic view of the domains of FtsB, FtsBhinf, FtsL, FtsLhinf, and the obtained swap
proteins BBhinfB and LLhinfL. Alignments of the E. coli and H. influenzae leucine zipper domains (LeuZip) swapped are shown with single-letter
abbreviations. The seven residues of each periodic repeat are referred to by the letters a to g. The d position is occupied by a conserved leucine
shown in bold. Conserved residues are indicated between the two sequences, and plus signs indicate conservative substitutions. (C) BACTH
analysis of T18 and T25 fusion proteins in the E. coli DHM1 strain (21). The genes encoding FtsLhinf and FtsBhinf were amplified by PCR from
H. influenzae chromosomal DNA, and the swap constructs LLhinfL and BBhinfB were constructed by overlapping PCR, cloned into the pKT25
and pUT18C vectors, and transformed into DHM1 (see the supplemental material). Relative ?-galactosidase activities in the E. coli DHM1 strain
coexpressing T18 and T25 fusion proteins were measured as described in the supplemental material. The levels of the ?-galactosidase activity in
the control cells expressing nonfused T25 and T18 fragments were below 1 relative unit.
VOL. 193, 2011NOTES 4991
suggest that the selective heterodimerization of the FtsL and
FtsB proteins in each species is partly specified by their LZ
Concluding remarks. Our present mutational analysis of the
key leucine residues from the LZ motifs of FtsL and FtsB
revealed the contribution of the hydrophobic interface be-
tween these motifs to the stable association of the FtsL and
FtsB proteins. The study of chimeric derivatives, LLhinfL and
BBhinfB, obtained by replacing the LZ motifs of E. coli FtsL
and FtsB with their corresponding motifs from the H. influen-
zae counterparts, further confirmed that the LZ motifs are
important determinants of the species-specific heterodimeriza-
tion of these two cell division components. Our results there-
fore demonstrate that the LZ motifs of FtsL and FtsB are
required for an optimal interaction between these two essential
proteins. Yet, additional regions of these proteins, especially
their transmembrane segments, are clearly contributing to the
stabilization of the FtsL/FtsB complex (5, 18). Furthermore,
the complexity and redundancy of interactions between the
divisomal proteins may also directly influence in vivo the
stability of individual components, as illustrated here by
the fact that FtsQ could efficiently stabilize the FtsBMor
We thank Agnes Ullmann for critical reading of the manuscript.
This work was supported by grant GMO-38922 of the National
Institute of General Medical Sciences, Bethesda, MD. J.B. is an Amer-
ican Cancer Society Professor. C.R. holds a Marie Curie Outgoing
International Fellowship of the European Community under contract
number MOIF-CT-2005-008977 and a Roux postdoctoral grant from
the Institut Pasteur.
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