From the Department of Infectious Disease,
Wyeth-Ayerst Research, Pearl River, New York 10965 and the
¶ Department of Molecular Biology and Microbiology, Case
Western Reserve University Medical School, Cleveland, Ohio
44106-4960
Received for publication, October 26, 2000, and in revised form, December 27, 2000
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ABSTRACT |
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The recruitment of ZipA to the septum by FtsZ is
an early, essential step in cell division in Escherichia
coli. We have used polymerase chain reaction-mediated random
mutagenesis in the yeast two-hybrid system to analyze this interaction
and have identified residues within a highly conserved sequence at the
C terminus of FtsZ as the ZipA binding site. A search for suppressors
of a mutation that causes a loss of interaction
(ftsZD373G) identified eight different changes
at two residues within this sequence. In vitro, wild type
FtsZ interacted with ZipA with a high affinity in an enzyme-linked
immunosorbent assay, whereas FtsZD373G failed to interact.
Two mutant proteins examined restored this interaction significantly.
In vivo, the alleles tested are significantly more toxic
than the wild type ftsZ and cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ
in the two-hybrid system, is sufficient for the interaction with FtsA
and ZipA. However, when the wild type sequence is compared with one
that encodes FtsZD373G, no interaction was seen with either
protein. Mutations surrounding Asp-373 differentially affected
the interactions of FtsZ with ZipA and FtsA, indicating that these
proteins bind the C terminus of FtsZ differently.
In bacteria, the study of cell division has defined many genes
active in the formation and cleavage of a septum (1-3). Currently, the
earliest known step in the development of the septum is the formation
of the Z-ring, a circular polymeric structure formed by the
tubulin-like protein FtsZ (4). The cell division machinery assembles on
the Z-ring in a sequential manner. Two proteins that act early in cell
division, and directly on the Z-ring, are FtsA and ZipA (5-10). The
structure of FtsA has been solved recently (11), and it supports
previous indications that it is similar to actin (12, 13). FtsA may
function by linking septum formation to peptidoglycan synthesis (8,
14). ZipA is an integral membrane protein that causes FtsZ fibers to
bundle in vitro (15, 16). ZipA is thought to stabilize the
FtsZ rings, in part because moderate overexpression of zipA
can suppress the ftsZ84 allele, which confers a defect in
Z-ring formation (16). The structure of the highly conserved C-terminal
fragment of ZipA has been solved by x-ray crystallography and NMR, and
it shows conservation to several RNA-binding proteins (17, 18).
Genetic analyses of cell division have identified many genes as
conditional alleles, but detailed genetic analysis describing an
individual protein·protein interaction is difficult, because many interacting proteins are involved, often simultaneously (19-21). Because of this, it is advantageous to develop a surrogate system that
allows the study of a protein·protein interaction through genetic
analysis (i.e. the study of a protein·protein interaction through the identification and characterization of mutations). The
two-hybrid system of yeast
(Y2H)1 is one such system,
because of the wide range of genetic techniques that exist for yeast
(22, 23) and because the two-hybrid system itself is a robust system
for characterizing protein·protein interactions in vivo
(24). Thus, it is a relatively simple extension of yeast genetic
analysis to examine a two-hybrid interaction genetically. Previous work
has indicated that the C terminus of FtsZ is important for the
interaction with both ZipA and FtsA (9, 10, 15, 21, 25). To better
define the interaction between FtsZ and ZipA, we have analyzed this
interaction in yeast genetically. Using a mutation in ftsZ
that reduces the interaction with zipA in the Y2H system, we
have searched for intragenic suppressors (additional mutations within
ftsZ that reverse the phenotype of the initial
loss-of-function mutation). This search has identified mutations within
a small segment of ftsZ that encodes a conserved sequence at
the C terminus. We have characterized the effect of these mutations on
the interactions with ZipA and FtsA and determined the effect of the
mutations on cell division.
Strains, Plasmids, and Oligonucleotides--
All strains and
plasmids used in this study are listed in Table 1s. Oligonucleotides
are listed in Table 2s. Both tables are published as supplemental
material in the online version of this paper.
Media and Reagents--
Yeast and bacterial media were prepared
by standard methods using materials readily available (22, 23). SC, YPD
and other general yeast media are described in these references. YNB,
BactoAgar, BactoTryptone, BactoPeptone, and yeast extract were
purchased from Difco. Amino acid mixtures (CSM-LUTH, CSM-AHT),
raffinose, glucose, and galactose were purchased from Bio101. Amino
acids, 5'-fluoroorotic acid (FOA), 4-methylumbelliferyl
Construction of Plasmids--
All oligonucleotides used in this
study are listed in Table 2s. FtsZ and ZipA were cloned by PCR
amplification of genomic DNA from Escherichia coli strain
MG1655. Wild type ftsZ cloned into pGAD424 was done by PCR
amplification of the ftsZ gene from pDR3 using the oligos
FtsZ-5' and FtsZ-3'. The amplified PCR product was digested with
MfeI and SalI and cloned into pGAD424 that was digested with EcoRI and BamHI. zipA
was amplified by PCR using oligos ZipA-5' and ZipA-3'. The ZipA-5'
oligo results in a PCR product that deletes the membrane-spanning
portion of the ZipA gene product. The resulting fragment was digested
with EcoRI and SalI and ligated into pLexA, to
generate plasmid pSH47. This fragment was subsequently excised and
cloned into pGAD424, which was digested identically to generate plasmid
pSH230. The plasmid pSH228 was constructed by PCR amplification of
plasmid pSH256 with oligos FtsZ-5'(311) and FtsZ-3'. The resulting
fragment was digested with EcoRI and SalI and
cloned into pAS2-1. pSH229 was constructed in an identical manner,
except that pSH41 was used as the template for the PCR reaction. pSH100
was constructed by PCR amplification of pAS1-ftsA, which was
generously provided by Sandy Silverman, with oligos FtsA-5' and ADHt.
The resulting fragment was digested with EcoRI and
SalI and ligated into pAS2-1. Plasmid pSH232 was constructed
by subcloning the ftsA gene into pGAD424, which had been
digested with EcoRI and SalI as well.
Construction of Yeast Strains--
Strain SHy9 was generated by
growing strain CG 1945 serially for two 10-ml overnight cultures, with
about a 105 cell inoculum each, and plating on SC plates
supplemented with 0.1% FOA, and colonies were allowed to grow for 5 days. Several colonies that grew were rechecked for all phenotypes,
including loss of GAL1p-lacZ reporter activity. Strains
SHy22 and SHy23 were generated by introducing pHO (kindly provided by
Kim Arndt) into strain SHy9 and growing a transformant in 10 ml of
SC-URA overnight. Cells were streaked onto a YPD plate and allowed to grow for 3 days. This plate was then replica printed onto an SC plate
supplemented with 0.1% FOA. Colonies from this plate were patched onto
a new YPD plate, grown overnight, and replica-printed onto an SPO
plate. This plate was incubated at room temperature for 24 h and
then at 30 °C for 5 days. Patches producing asci were then incubated
with Zymolyase, and spores were separated by the random spores
technique and plated on YPD (29). SHy22 and SHy23 are strains from
spores of the same patch and differ only by mating type.
PCR-mediated Mutagenesis and Selection of
Mutations--
Mutations in FtsZ were generated by PCR amplification
of pSH27 (pGAD424-ftsZD45N,D373G) using
Taq DNA polymerase and reaction conditions that favored the
incorporation of mutations. Oligonucleotides used as primers for
amplification were GAL4ad and ADHt. These primers annealed to the GAL4
activation domain and the ADH terminator regions, respectively, and
produced a PCR product that included about 300 bp of sequence on either
side of the ftsZ gene. These regions of homology allowed for
homologous recombination of the PCR fragment when cotransformed, with
pGADGH vector DNA that had been linearized by digestion with
EcoRI and BamHI, into SHy63 (30). Recombinants were selected by leucine prototrophy. In this work, cotransformation with 100 ng of plasmid DNA and PCR fragment (each) resulted in about
1000 colonies, whereas transformation by either the vector or the PCR
fragment alone resulted in zero to four colonies when plated onto LT
plates. The interactions between the mutagenized ftsZ gene
and zipA were tested by replica printing the transformants onto LHT plates supplemented with 0.5 mM AT. Colonies that
grew after 3 days when incubated at 37 °C were scored as hits. Each colony was grown as a 5-ml culture and prepared using the Qiagen Qiaprep Turbo 8 miniprep kit as described by the manufacturer, except
that 0.5 mg of Zymolyase was added per ml of buffer P1, and samples
were incubated for 30 min at 30 °C in this solution. These
preparations were used to transform E. coli strain
KC8 by electroporation, and colonies were selected by growth on M9
plates that were supplemented with 50 mg/liter ampicillin, but lacked leucine, as described by Golemis et al. (31). Two to four
colonies from each transformation plate were grown and prepared with
the Qiagen miniprep kit again, this time without modification to the manufacturer's instructions. DNA from these preparations were analyzed
by restriction analysis, and reconfirmation of the phenotypes was
achieved by transforming into SHy23 again and rescoring the AT resistance.
Site-directed Mutagenesis of Plasmids for Expression in Yeast and
Bacteria--
Mutations identified in the previous section were
further characterized by introducing the identified mutation into a
pGAD424-ftsZ construct. This was accomplished by
site-directed mutagenesis using the QuikChange site-directed
mutagenesis kit by Stratagene. Mutagenesis was performed as described
by the manufacturer. Oligonucleotides used in this work were: D373G/T,
D373G/B; D373S/T, D373S/B; D373G, P375L/T, D373G, P375L/B, as listed in
Table 2s. For the alanine-scanning mutations, site-directed changes
were introduced into pGAD-ftsZD373G (pSH-201).
The mutation that results in a change from D to G at position 373 also
results in a loss of the EcoRV restriction site. Each of the
oligo pairs encode a change that restores this restriction site, in
addition to the change at the codon to be changed to alanine. The
oligos used were: D370A/T, D370A/B; Y371A/T, Y371A/B; L372A/T, L372A/B;
F377A/T, F377A/B; L378A/, L378A/B; R379A/T, R379A/B; K380A/T, K380A/B;
Q381A/T, Q381A/B. Candidate clones were screened for the reacquisition
of the EcoRV site.
Purification of FtsZ and ZipA and Assay of the FtsZ·ZipA
Interaction in Vitro--
The wild type and mutant FtsZs were
expressed with the N-terminal biotin tag MAGGLNDIFEAQKIEWH (32) to
enable detection in an ELISA. The lysine in this sequence is
biotinylated in vivo by the E. coli enzyme BirA.
Plasmids were constructed by inserting the coding sequence for the
biotin tag between the NcoI and NdeI sites of
pET28 (Novagen) using the oligos BIOTAG/T and BIOTAG/B. In addition,
birA was amplified from the plasmid pBIOTRX-BirA (26) by PCR
using the oligos BirA 5' and BirA 3', digested with HinD III
and XhoI and ligated into the HindIII and
XhoI sites of the same vector as the biotin tag to give the
vector pETbio-birA. The genes ftsZ,
ftsZD373G, ftsZD373S, and
ftsZD373G,P375L were subcloned from the vectors
pDB312, pEG028, pSH187, or pSH189, respectively, into the
NdeI and HindIII sites of pETbio-birA to give
plasmids pEG045, pEG051, pEG052, and pEG053.
Biotin-FtsZ and its mutants were expressed in the E. coli
strain BL21(DE3)pLysS. Expression was induced with 1 mM
isopropyl-
Protein concentrations of biotin-FtsZ and its mutants were determined
by the Bradford method. The extent of incorporation of biotin was
determined by measuring the displacement of
2-(4'-hydroxyazobenzene)benzoic acid from avidin. In short, 40 µl of
protein sample or buffer was mixed with 360 µl of 0.5 mg/ml avidin
and 0.3 mM 2-(4'-hydroxyazobenzene)benzoic acid in 100 mM sodium phosphate, 150 mM NaCl, pH 7.2. The
decrease in absorbance at 500 nM was measured, and the
concentration of biotin was determined using
ZipA-(23-328) was overexpressed from the plasmid pDB348 in
BL21(DE3)plysS. Expression was induced as for biotin-FtsZ, above, and
the cells were centrifuged, resuspended, and stored similarly. At the
time of purification, the cells were thawed, phenylmethylsulfonyl fluoride was added to 1 mM, and the cells were lysed by
passage through a French press. The cell extract was clarified by
centrifugation at 100,000 × g for 1 h, and
ZipA-(23-328) was precipitated by adding ammonium sulfate to 35%
saturation. The ammonium sulfate pellet was dissolved in buffer A and
dialyzed against buffer A overnight. ZipA-(23-328) was purified to
homogeneity by passage over a Mono-Q column (Amersham Pharmacia
Biotech) and elution with a 50-230 mM gradient of KCl in
buffer A. The protein concentration of ZipA-(23-328) was determined as
described according to Gill and von Hippel (34).
The interaction between ZipA-(23-328) and biotin-FtsZ and its mutants
was assayed in an ELISA format. ZipA-(23-328) was immobilized in the
wells of an Immulon 4HBX 96-well plate in 50 mM Tris, pH 8.5, 100 mM NaCl at 1 µg/ml overnight at 4 °C. Unbound
ZipA-(23-328) was removed, and the wells were blocked with blocking
buffer (0.2% bovine serum albumin in PBS-T (10 mM
Na2HPO4, 1.8 mM
KH2PO4, pH 7.5, 140 mM NaCl, 2.7 mM KCl, 0.05% Tween 20)). After two washes with PBS-T,
biotin-FtsZ and its mutants were added at various concentrations in
blocking buffer for 1 h at room temperature. Unbound FtsZ was
removed, and the wells were washed three times with PBS-T. Next, 0.1 µg/ml streptavidin-horseradish peroxidase conjugate in blocking
buffer was added and incubated at room temperature for 1 h. The
wells were washed four times after the removal of the conjugate. The
horseradish peroxidase substrate o-phenylenediamine was then
added in sodium phosphate-citric acid buffer, color development was
stopped after a few minutes with 1.3 N
H2SO4, and the absorbance at 490 nm was measured.
Cell Biology Methods--
E. coli morphology was
determined by phase contrast microscopy of E. coli cells as
described previously (5). Experimental conditions are described in the
legends for Tables I and
II.
A Two-hybrid System That Allows for the Selection of Mutations in
ftsZ, which Affect the FtsZ·ZipA Interaction--
Our interest in
developing a genetic system to characterize the FtsZ·ZipA interaction
was piqued by the observation that a mutation in ftsZ
resulted in a reduced interaction with zipA, when studied in
the yeast two-hybrid system. This observation was made when one of
several clones derived by PCR amplification of E. coli
genomic DNA was subcloned into the two standard two-hybrid systems.
When ftsZN45D,D373G was expressed as a fusion to
the B42 activation domain, an interaction was seen with zipA
expressed as a fusion to the LexA DNA binding domain. This interaction
could clearly be scored in standard analyses, such as on indicator
plates containing 5-bromo-4-chloro-3-indolyl
The lack of growth in the galactose system provided a clear
strategy for determining which residues in FtsZ facilitate binding to
ZipA, through the selection of intragenic suppressors. Selection of
suppressors that restored the FtsZ·ZipA interaction was achieved by
PCR mutagenesis of the whole ftsZ gene as cloned into
pGAD424, using primers that annealed to the GAL4 activation
domain fragment and the ADH terminator. The primers allowed
~300-bp extensions to both ends of the ftsZ gene. These
extensions provided regions of homology that allowed the PCR products
to be cloned by recombination (Fig. 2A) (30). The PCR
products were transformed directly into strain SHy63 (a derivative of
the Gal-Y2H strain CG1945 that had been previously transformed with
pAS2-1-zipA), with the pGADGH vector that had been
linearized, and is therefore not stable in yeast unless it has been
repaired. Repair could be achieved by homologous recombination with the
ends of the PCR products containing portions of the GAL4
activation domain gene, and the ADHt terminator. Transformation into strain SHy63 allowed the selection of recombinant plasmids expressing ftsZ alleles. Alleles interacting with
the Gal4bd-zipA fusion were identified through the
activation of the GAL1p-HIS3 reporter. 73 colonies grew on
plates that lacked histidine and were supplemented with 1 mM aminotriazole, two of which are shown in Fig.
2B. Plasmids were recovered and retested in yeast, and 12 plasmids were purified that could be recovered, had normal restriction
analysis patterns, and conferred plasmid-dependent phenotypes (histidine prototrophy only when introduced into a strain
that carried pAS2-1-zipA). The ftsZ genes in
these 12 plasmids were sequenced to identify any mutations. Eight of
the 12 plasmids contain mutations in the conserved C terminus region.
The remaining four plasmids do not contain mutations that result in
amino acid changes in FtsZ are presumed to contain mutations that
affect the copy number of the plasmid or the expression of the
Gal4·FtsZ hybrid protein and have not been characterized further. The
eight mutations identify five different residue changes from the
original plasmid. Two mutations are reversions to the wild type
aspartate residue at position 373, two mutations change the Asp-373
residue to serine, and one changes this glycine to cysteine. The
remaining suppressors change the highly conserved proline residue at
position 375 to leucine (twice) and to serine. These mutations are
indicated in Fig. 3. The residues that
comprise the C terminus of the E. coli FtsZ are shown.
Residues in capital letters show conservation among FtsZ
proteins from prokaryotes and plants. The mutations identified in yeast
that are critical for the interaction of FtsZ with ZipA map within this
sequence, and no mutations from other regions of ftsZ were
identified, indicating that these residues may comprise the principal
region of interaction with ZipA.
The suggestion that these mutations have significant effects on the
interaction of FtsZ with ZipA was confirmed by introducing some of them
into an unmutagenized pGAD424-ftsZ plasmid, and rechecking the phenotypes. Wild type ftsZ was compared with
ftsZD373G, ftsZD373S, and
ftsZD373G, P375L. We checked the interaction
with zipA and ftsA in the galactose Y2H
system (Fig. 4). The latter interaction
was examined, because other work has suggested that FtsA also interacts
with FtsZ at its C terminus, and we were therefore interested in
characterizing this interaction (10, 21, 35). As can be seen in Fig. 4, the FtsZ·FtsA interaction is also highly sensitive to mutations in
the C terminus of FtsZ. All strains tested show good growth on plates
supplemented with histidine (Fig. 4A). The interaction of
ZipA with FtsZ is best scored on plates containing 0.5 mM
AT (Fig. 4B), where robust growth is seen for the wild type
FtsZ, and for the two suppressors identified in Fig. 2. Expression of the GAL4-zipA fusion confers a higher background than either
the vector control or the GAL4-ftsA fusion, giving growth on
plates without AT, regardless of which GAL4AD
construct is expressed. In the case of ftsA, the interaction
is more sensitive, and its interactions with ftsZ can be
scored on plates lacking histidine and AT (Fig. 4B). In this
case, ftsA interacts well with wild type ftsZ,
and with the ftsZD373S allele. It does not
interact with either allele that changes the Asp-373 residue to
glycine.
Further characterization of the interaction between ZipA and FtsZ was
performed in vitro with purified proteins. ZipA-(23-328), the soluble form of the protein, which lacks the N-terminal
membrane-spanning domain, and FtsZ proteins, expressed from the alleles
examined in Fig. 3, were purified from E. coli to
homogeneity. The interactions were tested in an ELISA, as shown in Fig.
5. Wild type FtsZ shows a high affinity
for ZipA, having an apparent dissociation constant of 0.19 (±0.009)
µM. The affinity of the FtsZD373G mutant for
ZipA was too low to be quantified in this assay, despite the protein
being otherwise well behaved, indicating that the mutation has a
significant impact on the interaction of these two proteins. Two
suppressors were analyzed as well, FtsZD373S and
FtsZD373G, P375L, and were shown to have dissociation
constants of 6.2 (± 0.9) µM and 1.3 (± 0.2)
µM, respectively. Both proteins show greatly improved
interactions with ZipA, although neither protein interacts with
ZipA as well as wild type FtsZ.
FtsA Also Binds to the Conserved Carboxyl Terminus of FtsZ, but in
a Manner Different from ZipA--
The results in Fig. 4, as well as
work by others (9, 10, 15, 21, 35), strongly implicate the C terminus
of FtsZ as playing a critical role in the interaction of FtsA, in
addition to ZipA. We were interested in examining the role of
additional residues in the conserved C terminus of FtsZ in binding to
FtsA as well as ZipA. In Fig. 6, we
examined the role of the C terminus in binding to ZipA and FtsA in the
Y2H system. In this experiment, we expressed a segment of the
ftsZ gene, which encodes the last 70 residues of FtsZ as a
fusion to the GAL4bd, rather than to the
GAL4ad, as has been done in previous
experiments. The ftsZD373G mutation, expressed
in a similar construct, clearly indicates that this residue plays a key
role in the interaction of both fusions assayed.
Next, we sought to determine whether ZipA and FtsA interact with
identical residues in the FtsZ C terminus. Eight additional residues
within this conserved sequence were mutated individually to alanine.
Strains expressing derivatives of pGAD424-ftsZ that have
specific residues changed to alanine are indicated in Fig. 7. The strains were characterized for
histidine prototrophy and for Phenotypic Consequences of Mutations That Change Residues in the
FtsZ C Terminus--
The mutations described above were assayed in
E. coli to determine whether there were any biological
consequences associated with them. The mutations were introduced into
pDR3, a plasmid that expresses ftsZ under the control of the
lac promoter, and can complement a ftsZ deletion
when induced by IPTG. Two assays were performed. In the first,
ftsZ alleles expressed under a regulated promoter were
characterized for their ability to complement an ftsZ
deletion. The results are presented in Table I. pDR3 efficiently complements in the presence of IPTG, whereas none of the mutants can.
Additional effects can be seen in Table II, where the expression of
ftsZ from the same plasmids as those in Table I are examined for dominant effects. Expression of extrachromosomal ftsZ is
toxic at high levels, as can be seen when a strain carrying pDR3 is exposed to high concentrations of IPTG, which expresses the plasmid copy of the FtsZ gene to high levels.
Dominant effects can be seen for all of the mutated alleles, with
strong effects seen with the ftsZD373G allele
and the ftsZD373G,P375L allele. In these cases,
an Sep Recently, the bacterial cell division proteins FtsZ, ZipA, and
FtsA have been structurally characterized, as well as the ZipA·FtsZ interaction (11, 17, 18, 36). In addition to these structural characterizations, functional and biological studies, such as those
described in this report, will allow us to understand how biologically
important residues function in these interactions. Results presented
here show that several residues within the conserved C terminus of FtsZ
play essential roles in cell division, and minor changes within this
sequence can have lethal consequences.
The results presented in this study that characterize the role of
individual residues in the biological context of the Y2H system are in
general agreement with the in vitro characterization of
Mosyak et al. (17). One area where differences were seen is
the first group of conserved residues: Asp-370, Tyr-371, and Leu-372.
All three residues were shown to contribute important binding energy in
the BIAcore system, but only the Y371A change was shown to be important
in the Y2H system. The L372A change functioned well in the Y2H system,
despite being the most important of the three in vitro. It
is possible that the FtsZ binding pocket of ZipA can accommodate the
D370A and L372A changes in the Y2H system more effectively than it can
in vitro or that it can accommodate the Y371A change in the
in vitro system better than in the Y2H system. Two
mutations, L372A and R379A, affected the interaction with
ftsA to a greater extent than the interaction with
zipA. The severe effect of the R379A change is especially
interesting, because structural data show that the arginine residue is
solvent-exposed when bound by ZipA, which is consistent with the modest
effect with zipA in yeast. These results clearly indicate
that, although both proteins bind the same C terminus core of FtsZ,
they bind to different residues within this core. Residues that were
identified as playing a major role in the interaction in
vitro showed similarly significant roles in the experiments
presented here.
This work was initiated through an exhaustive search for suppressors of
the loss-of-function allele, ftsZD373G. We
identified only residues in the conserved C terminus, despite the fact
that the entire ftsZ gene was subjected to mutagenic PCR. As
these results were first obtained, it was surprising that second-site
intragenic suppressors were obtained at only one residue, Pro-375. If
this conserved segment was the site of the interaction with ZipA, then
one might expect that mutations that increase the affinity of ZipA for
the D373G mutant of FtsZ would be restricted to this region, but the
presence of suppressors at a single residue was not anticipated.
Structural studies have shed some light on our results (17, 18).
Although the Asp-373 residue plays a critical role in the interaction
between FtsZ and ZipA, it is not directly involved in the interaction
with ZipA per se. Rather, it plays a critical role in
defining the structure of the residues that contact ZipA significantly.
This is illustrated in Fig. 8. This
figure shows the FtsZ peptide bound by the C-terminal domain of ZipA,
as determined by x-ray crystallography, and is depicted using the
program RIBBONS (37). The Asp-373 side chain contributes a hydrogen
bond to the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactoside, and aminotriazole were purchased
from Sigma Chemical Co. Zymolyase was purchased from ICN Biologicals.
-D-thiogalactopyranoside (IPTG) once
the A600 of the culture was between 0.5 and 1.0. At the same time, D-biotin was added to a final
concentration of 0.1 mM. Cells were incubated at 37 °C
for another 2-3 h, centrifuged, and resuspended in buffer A (50 mM Tris, pH 7.9, 50 mM KCl, 1 mM
EDTA, and 10% glycerol) and stored at
70 °C. The proteins were
then purified according to a previous report (33).
500 × 10
3 = 34. The biotin-tagged FtsZs were between 50% and
75% biotinylated.
Complementation of a ftsZ deletion by alleles of ftsZ expressed from a
regulated promoter
trpE
trpA
tna
recA::Tn10
ftsZo/repAts ftsZl], and transformants
were grown overnight at 30 °C in LB + Ap (50 µg/ml) + Cam (50 µg/ml) + glucose (0.1%). Cultures were diluted in LB to
A600 = 1 × 10
7, and 0.1-ml aliquots were spread
on four plates. One plate containing LB + Ap (50 µg/ml) + glucose (0.2%) was incubated at 30 °C. The other plates were
incubated at 42 °C. These plates contained LB + Ap (50 µg/ml)
supplemented with no. 5, or 10 mM IPTG. For each plate, the
number of colony forming units was determined. Plasmids used in this
experiment were: vector, pMLB1113; wild type ftsZ, pDR3;
ftsZD373G, pSH179; ftsZD373S,
pSH181; ftsZD373G,P375L, pSH183.
Dominant effects of ftsZ mutations expressed in E. coli
trpE
trpA
tna
recA::Tn10], and
transformants were grown overnight at 37 °C in LB + Ap (50 µg/ml) + glucose (0.1%). Cultures were diluted 200-fold in
LB + Ap (50 µg/ml) supplemented with the indicated concentration
of IPTG, and growth was continued at 37 °C for 4-5 h until
A600 = 0.8-1.0. Division phenotypes were determined phase
microscopy. Plasmids used in this experiment are described in Table I.
-Galactosidase Assay--
Cultures to be tested were grown
for 36 h in 5 ml of SC-Leu-Trp media. New cultures were inoculated
with 100 ml of the overnight cultures, and the new cultures were grown
for 16 h. Cells densities were between 0.8 and 1.0 A600 for these strains. Samples of these cultures were assayed for LacZ activity in quadruplicate, in a 96-well
microtiter plate (100 µl per well). Samples were mixed with 100 µl
of lysis buffer and substrate (40 µl of Promega cell lysis buffer, 40 µl of 0.125 mg/ml 4-methylumbelliferyl
-D-galactoside (Sigma), and 20 µl of 10×
-galactosidase assay salts). Samples were incubated at 30 °C for
4-8 h with shaking and read on a Victor II fluorescence plate reader
from Wallac. Fluorescence intensity increased with time, and
after 8 h, negative control wells showed about 400 units, whereas
positive control wells for the ZipA·FtsZ interaction showed about
50,000 units and for the FtsA·FtsZ interaction showed about 10,000 units.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (X-gal), and by leucine
prototrophy, but was significantly less robust than a fusion to wild
type ftsZ (Fig. 1). No
interaction was seen when the mutated ftsZ allele was
examined in the galactose system (Fig.
2B, and results not shown). An
examination of the mutations introduced during cloning suggested that
one of the mutations could be important for the FtsZ·ZipA
interaction. The first change was N45D, which has been characterized by
Wang et al. (35) as one that affects the GTPase activity of
FtsZ. This change was discounted as the cause of the altered
interaction with ZipA by several experiments (Ref. 15, and results not
shown). This evidence includes the observation that ZipA binding to
FtsZ is not affected by guanine nucleotides and that deletion analysis
of FtsZ had already shown that the GTPase domain of FtsZ was not
involved in binding to ZipA. The other mutation, D373G, resulted in a
change in a highly conserved region of the C terminus of FtsZ
(discussed below). At the time this project began, the role of this
region in cell division had not been characterized. This mutation
provided us with an opportunity to study how a mutation with diminished
function could be used to characterize this interaction.
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Fig. 1.
The interaction of ftsZ with
zipA in the yeast two-hybrid system is specific and
sensitive to mutations in ftsZ. Diploid
strains were constructed by mating EGY48 containing either pLexA
or pSH47 (pLexA-zipA) with YM4271 containing pB42, pSH256
(pB42-ftsZ, wild type), or pSH48 (pB42-ftsZ
D39N,D373G). Overnight cultures were spotted into a
microtiter plate containing 100 µl of media per well, and 5-µl
spots were applied onto plates using a pin arrayer, as indicated in the
figure. Plates were incubated at 30 °C for 4 days.
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Fig. 2.
Isolation of intragenic suppressors of the
ftsZD373G mutation. A,
mutations in ftsZ were generated by mutagenic PCR
amplification of the ftsZD373G allele as a
construct in pGAD424. Primers for the amplification were to sequences
about 300 bp away from the multiple cloning site of the vector,
allowing for cloning by in vivo recombination and expression
of clones carrying mutations that restored the interaction of
ftsZ with zipA. B, growth phenotypes
of pGADGH-ftsZ plasmids recovered from the screen. Plasmids
were transformed into yeast strain CG1945, along with either plasmid
pAS2-1 or plasmid pSH227 (pAS2-1-zipA), as indicated, grown
overnight, and spotted onto the indicated plates.
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Fig. 3.
Summary of mutations isolated by PCR
mutagenesis in the two-hybrid system. The FtsZ C terminus is shown
as both the wild type sequence (top line), and with the D to
G mutation that was encoded by the template DNA for this work
(second line). Suppressors isolated from this DNA are
indicated in the third line. Residues Asp-373 to Pro-375 that comprise
the signature DIP sequence are underlined. FtsZ residues are
numbered. Conserved residues are capitalized.
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Fig. 4.
Interaction of intragenic suppressors of the
ftsZD373G mutation with zipA
and ftsA in the two-hybrid system. Yeast
diploid strains resulting from crosses of yeast strain SHy22 containing
pAS2-1, pSH227 (pAS2-1-zipA), or pSH 100 (pAS2-1-ftsA) with yeast strain SHy23 strains containing
pGAD424-ftsZ-based plasmids as indicated in the figure.
Diploid strains for testing are grown overnight and spotted, as
described in Fig. 1, onto the plates indicated in the figure.
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Fig. 5.
Determination of the dissociation constants
for the binding of FtsZ and FtsZ mutants by ZipA. The interaction
was assayed as described under "Experimental Procedures," and the
data ( , biotin-FtsZ;
, biotin-FtsZD373G, P375L;
,
biotin-FtsZD373S; and
, biotin-FtsZD373G)
were fit by linear regression with a steady-state affinity model. Each
data point is an average of three repeats.
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Fig. 6.
zipA and ftsA
interact with the C terminus of ftsZ in the
yeast two-hybrid system. Strains were constructed as described in
Fig. 4 using strain CG1945 instead of strain SHy22 for the bait
plasmids (pAS2-1, pSH228 (pAS2-1-ftsZ-(311-383)), and
pSH229 (pAS2-1-ftsZD373G-(311-383)). Prey
plasmids (pGAD424, pSH230 (pGAD424-zipA), and pSH232
(pGAD424-ftsA)) were transformed into strain SHy23 to obtain
strains for mating. Controls were performed as described in Fig. 4, but
are omitted from the figure for clarity. Duplicate spots
represent overnight cultures of two independent transformants.
-galactosidase activity, as shown in
the figure. In this experiment, strain CG1945 was used for the bait
plasmids. In these diploids, background growth of the strains
containing pAS2-1-zipA were more comparable to that of the
strains containing pAS2-1-ftsA, so only the SC-LHT plate is
shown. Robust growth on plates containing 0.5 mM AT was
only seen with the zipA·ftsZ interactions (data not
shown).
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Fig. 7.
Comparison of the interaction of
ftsA and zipA with alanine-scanning
mutations in other conserved residues of the ftsZ C
terminus. Strains were constructed as described in Fig. 6.
phenotype is seen at substantially lower
concentrations of IPTG. The same is true for the
ftsZD373S allele, which has a more modest
phenotype relative to the other mutations in this assay as well, but is
still substantially more toxic than the wild type gene expressed on a
plasmid. Thus, a comparison of the ftsZ alleles with the
wild type ftsZ indicates that all of the mutations examined
in Tables I and II show profound cell division defects.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carbon backbone, which helps define the transition from
-sheet structure to
-helix. The change to glycine introduces a
significant structural change, including the removal of this critical
hydrogen bond. The Pro-375 residue also plays a structural role in
defining the ZipA binding site. The residues that directly interact
with ZipA are interspersed through the sequence that includes the
Asp-373 and Pro-375 residues, in particular, residues Ile-374, Phe-377,
and Leu-378 (17). Therefore, the structure of the FtsZ C terminus is
critical because of the number of interactions that occur along the
-carbon chain of FtsZ (17, 18). The best explanation that can be
offered from the structural data is that the D373G change perturbs the
orientation of the interacting residues, and this perturbation is
alleviated by the P375L mutation. In the inset of Fig. 8,
the D373G and P375L mutations are modeled into the structure. The
inset shows where the hydrogen bond is lost, and where the
leucine side chain would interact with ZipA. Further structural studies
will be required to determine whether the P375L mutation provides an
additional hydrophobic interaction with ZipA or whether it allows
hydrophobic capping of the FtsZ peptide to restore the conformation of
the interacting residues. Finally, although the number of mutations
that can negatively affect an interaction is fairly large, the number
of possible changes that can increase the strength of the
interaction seems to be small.
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Fig. 8.
Structure of the FtsZ·ZipA
complex. The interacting residues of FtsZ are indicated in
blue, with the Asp-373 and Pro-375 side chains shown. The
ZipA structure is shown in green. Numbers
indicate residues of the FtsZ peptide. The hydrogen bond between
Asp-373 and the main chain of FtsZ is indicated by the white
dashed line. In the inset, the D373G and P375L changes
are modeled into the ZipA·FtsZ structure. Representations were done
using the program RIBBONS (37).
Although none of the recent studies can conclude that the conserved C terminus residues of FtsZ comprise the complete target sequence for ZipA, it is certainly true that the region plays a critical role in the interaction with ZipA. The interaction of the FtsZ C terminus with ZipA has been characterized extensively (5, 9, 17, 18, 21). Additionally, the results of Fig. 6 indicate the C-terminal tail of FtsZ is sufficient for the interaction of FtsZ with both ZipA and FtsA. The portion of FtsZ encoded by the Y2H fusion plasmid in this figure consists of the last 72 residues of FtsZ. The interaction of ZipA with a MBP·FtsZ-(311-383) fusion protein, with the wild type protein, and the 17-residue peptide are all similar, as measured in a BIAcore biosensor assay.2 Therefore, it is possible to conclude that the mutations recovered in this study were limited to the conserved residues of the FtsZ C terminus, because this region defines the principal ZipA interaction site. Thus, we have met a goal of this study, which was to use a random genetic approach to help define a protein·protein interaction.
The suppressors identified in this study provide information about the sensitivity of the cell division machinery to changes in the interactions of its components. The FtsZD373G allele has profound effects on cell division and viability, as well as conferring on the mutant protein a greatly reduced affinity for ZipA and for FtsA. For these proteins, these results are consistent with the importance of their proper interaction with FtsZ. The suppressors show that these interactions are extremely sensitive. Neither of the alleles characterized in detail in this study (FtsZD373G, P375L or FtsZD373S) complement a deletion. In the case of the double mutation, this is less informative about the relation between affinity for ZipA and function, because it shows a dramatically reduced affinity for FtsA, which complicates our determination of why this mutation fails to complement. The other allele, ftsZD373S causes more modest changes on the interaction with ftsA. No difference from wild type could be seen between ftsA and ftsZD373S by Y2H assay, indicating that any change is relatively minor. The data, including Y2H and the in vitro analysis in Fig. 5, suggests that it is more likely that this allele fails to complement because of altered interactions with zipA. Although it is possible that another protein also interacts with FtsZ at this site, the simplest conclusion to draw at this time is that these proteins cannot tolerate even moderate changes in their interactions.
If the interaction characterized in this study represents an important antibacterial target, it is necessary to show that the interaction between ZipA and FtsZ is not just essential but is very sensitive to interference. Although it is true that many protein·protein interactions are essential, their identification as promising antibacterial targets depends on the interaction being sensitive to interference. In some cases, inhibition of expression or activity by 50% can be lethal. For many essential genes, reducing expression by 95% or more can result in no observable defect. Several genes have been characterized that are functional when they have activity at 1%, or less, of their wild type levels. Classic examples include nonsense mutations, when their phenotypes can be alleviated by suppressor tRNAs (38, 39). The strength of a protein·protein interaction, or an activity, as a pharmaceutical target can be evaluated by such data. Specifically, if reducing a protein·protein interaction moderately (50-80%) has phenotypic consequences, then it could be regarded as a strong pharmaceutical target. If reducing the interaction by 100-fold or greater is required to inhibit growth, it may be problematic to find a drug that can achieve this level of inhibition through its specific activity and pharmacokinetics, in a true in vivo situation. One of the reasons that cell division is considered an important area of antibacterial research is that many of its steps are very tightly controlled, and are very sensitive to changes in expression levels. Cell division is sensitive to changes of 2- to 4-fold in the expression of ftsZ, zipA, and other genes (3, 6, 40). If the FtsZ·ZipA interaction itself is as sensitive, then this interaction has potential as a target. The results presented here argue that this is the case.
It is clear that the yeast two-hybrid system is a powerful system for
the study of protein·protein interactions (24, 41-45). In this
report, we have taken advantage of commonly used techniques for
classical genetic analysis in yeast (i.e. the
characterization of gene function through the identification of
mutations) and applied them to a protein·protein interaction of
E. coli. The need for such methods rests on the observation
that most important cellular processes (both eukaryotic and
prokaryotic) are as dependent on protein·protein interactions as they
are on enzymological functions. Although inhibition of enzyme function
is well understood as a means of developing therapeutics, developing
compounds that function through the inhibition of protein·protein
interactions is much less well understood (46). Two clear examples of
drugs that inhibit a protein·protein interaction are that of FK506,
which inhibits the interaction of type I transforming growth factor- receptors with FKBP12 (47), and the peptidomimetic compound BILD 1263, which inhibits the interaction of the herpes simplex virus
ribonucleotide reductase subunits (48). The polymerization of
tubulin by taxol is another interaction affected by a chemotherapeutic, but in this case taxol functions by stabilizing the tubulin dimer associations (49, 50). Protein·protein interactions may comprise large surface areas, and those that do would be much less favorable as
drug targets. Applying the yeast two-hybrid system as a vehicle for
yeast genetic analysis provides a fairly rapid and general method for
determining the nature of a protein·protein interaction.
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ACKNOWLEDGEMENTS |
---|
We thank Eric Beer, Jie Wu, and David Fruhling for excellent DNA sequencing and analysis; Lidia Mosyak and Will Somers for helpful discussions; and Steve Projan and David Shlaes for continuous support. We thank L. Mosyak for providing Fig. 8.
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FOOTNOTES |
---|
* Part of this work was supported by NIH grant GM-57059 (to P. de B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Tables 1s and 2s.
¶ To whom correspondence should be addressed: Dept. of Infectious Disease, 205/277, 401 N. Middletown Rd., Pearl River, NY 10965. Tel.: 845-732-3683; Fax: 845-732-2480; E-mail: haneysa@war.wyeth.com.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M009810200
2 E. Glasfeld, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
Y2H, yeast
two-hybrid system;
FOA, 5'-fluoroorotic acid;
YPD, yeast-peptone
media with glucose;
SC, synthetic complete media, YNB, yeast nitrogen
base;
AT, 3-amino triazole;
bp, base pair(s);
ELISA, enzyme-linked
immunosorbent assay;
IPTG, isopropyl--D-thiogalactopyranoside.
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