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Journal of Bacteriology, September 2000, p. 5267-5270, Vol. 182, No. 18
Department of Microbiology and Immunology,
University of Rochester Medical Center, Rochester, New York 14642
Received 28 March 2000/Accepted 27 June 2000
Recently, M. Dmitrova et al. (Mol. Gen. Genet. 257:205-212, 1998)
described a LexA-based genetic system to monitor protein-protein interactions in an Escherichia coli background. However,
the plasmids used in this system, pMS604 and pDP804, were not readily
amenable for general use. In this report, we describe modifications of both plasmids that allow fragments of DNA to be fused to either vector
in any reading frame. Homodimerization and heterodimerization of
full-length proteins involved in polysialic acid synthesis in E. coli K1, as well as heterodimerization between a full-length protein and a protein fragment, demonstrate the usefulness of the
modified plasmids for investigating bacterial protein-protein interactions in vivo.
The K1 capsule of Escherichia
coli is a linear homopolymer of The genes for the biosynthesis and transport of the K1 capsule are
encoded on the 17-kb kps gene cluster, which is functionally divided into three regions (4, 21, 23). The genes in regions 1 and 3, designated kps, are conserved among E. coli strains that synthesize serologically distinct capsules and
are involved with transport of the polymer to the bacterial cell
surface (4, 21, 23). In contrast, the genes in region 2 are
unique to a given capsular type. In E. coli K1, region 2 encodes the neuDBACES genes, which direct the synthesis,
activation, and polymerization of NeuNAc (4).
Biosynthesis of polySia in E. coli K1 involves the
NeuB-catalyzed condensation of N-acetylmannosamine (ManNAc)
and phosphoenolpyruvate to form NeuNAc (20). ManNAc is
provided by the neuC gene product, a UDP-GlcNAc
2-epimerase (D. A. Daines, W. F. Vann, D. O. Chaffin, C. E. Rubens, and R. P. Silver, submitted for
publication). NeuD is also required for NeuNAc synthesis, although its
enzymatic role remains an enigma (5a).
Activation of NeuNAc is performed by NeuA, which adds a
nucleotide monophosphate to the sugar to form CMP-NeuNAc
(19). NeuS is the sialyltransferase, polymerizing
activated CMP-NeuNAc to polySia (16).
The current view of polymer synthesis and assembly assumes that the
components of the kps cluster form a hetero-oligomeric protein complex (4, 13). Identification and characterization of the protein-protein interactions involved in this complex are crucial to our understanding of the mechanism of polymer synthesis and
transport. Recently, a LexA-based genetic system for studying protein-protein interactions in an E. coli background was
reported (6). The LexA repressor is an important component
in the regulation of SOS response genes (14). The protein
consists of two domains, a DNA-binding domain (DBD) and a dimerization
domain. Although a truncated LexA consisting of only the DBD can
recognize its operator sequence, it is functional as a transcriptional
repressor only in dimeric form. Other domains can be fused in
frame to the DBD and will restore the repressor's function if
these domains interact. The LexA-based system described by
Dmitrova et al. (6) consists of two compatible plasmids
carrying either a wild-type (pMS604) or a mutant (pDP804)
lexA DBD gene fragment. The mutant LexA DBD contains three
amino acid changes that allow it to recognize a mutant operator. The
plasmids were used in two E. coli reporter strains, SU101
and SU202. SU101 has a wild-type LexA operator sequence upstream of a
lacZ reporter gene expressed by the sulA promoter
engineered in the chromosome, while SU202 has the same reporter system
but is controlled by a hybrid LexA operator sequence (6).
Since sulA is one of the most repressed genes of the SOS response, LexA binds the sulA operator more tightly than the
other natural SOS operators (14). A wild-type LexA fusion
homodimer recognizes the wild-type operator in SU101 and represses
reporter gene transcription, but only a heterodimer of a wild-type and a mutant LexA DBD fusion can recognize the hybrid operator in SU202.
This allowed both homo- and hetero-association of protein fusions to be
monitored. The system was tested for heterodimerization using
the leucine zipper domains of the eukaryotic proteins Jun (pDP804) and Fos (pMS604). Although the focus of the study was protein
heterodimerization, Dmitrova et al. (6) also analyzed homodimerization by fusing a Fos zipper region mutated so that it could
interact with itself to the wild-type LexA DBD.
Plasmids pMS604 and pDP804 carry the Fos and Jun leucine zipper
domains, respectively, and were not readily amenable for general use.
In this report we describe modifications of both plasmids that allow
fragments of DNA to be fused to either vector in any reading frame. We
demonstrate the usefulness of the modified plasmids for investigating
bacterial protein-protein interactions in vivo by demonstrating
homodimerization of full-length NeuD, NeuA, NeuC, and NeuB as well
as heterodimerization between NeuD and NeuB.
Modification of pDP804 and pMS604.
Plasmid pDP804 carries the
p15A origin of replication and an ampicillin resistance gene. Plasmid
pMS604 has a ColE1 origin of replication and a tetracycline resistance
gene (6). In each plasmid, a lac promoter
controls expression of the gene encoding the wild-type or mutant LexA
DBD fused to the leucine zipper region of the Fos or Jun protein,
respectively, and is therefore inducible by
isopropyl-
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Evidence for Multimerization of Neu Proteins
Involved in Polysialic Acid Synthesis in Escherichia coli
K1 Using Improved LexA-Based Vectors
and
ABSTRACT
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-(2,8)-linked
N-acetylneuraminic acid (NeuNAc; sialic acid) (15,
17). The capsule is identical to the polysialic acid (polySia)
found on human embryonic nerve cell adhesion molecules and is poorly
immunogenic due to the molecular mimicry of host structures
(17). Surface-displayed sialic acid also confers resistance
to the alternative complement pathway, allowing E. coli to
establish extraintestinal infections such as neonatal septicemia and
meningitis (15, 17).
-D-thiogalactopyranoside (IPTG)
(6). To remove the Fos leucine zipper fusion, pMS604 was
digested with the restriction endonucleases PinAI
(AgeI) and XhoI and gel purified by standard procedures (2). Plasmid pDP804 was subjected to
identical procedures, except that the restriction endonucleases were
BssHII and BamHI. PCR-generated fragments of the
multiple cloning sites (MCS) from the vectors pTrcHisA, pTrcHisB, and
pTrcHisC (Invitrogen, Carlsbad, Calif.) with compatible restriction
sites were ligated to the purified vector DNA. The six plasmids created
allow fragments of DNA to be fused to either the wild-type or the
mutant LexA DBD in any of three reading frames (Fig.
1). The pMS604 (wild type)-based plasmids
are pSR658, pSR660, and pSR662, carrying the MCS of pTrcHisA, -B, and
-C, respectively. The pDP804 (mutant)-based plasmids are pSR659,
pSR661, and pSR663, which carry MCS-A, MCS-B, and MCS-C, respectively.
Details of constructions and sequences of the vectors can be obtained
from R. P. Silver. Using the modified plasmids, an entire gene or
region of interest can be fused to the wild-type LexA DBD, and
homodimerization can be monitored by lacZ reporter gene
transcriptional repression in SU101 via -galactosidase activity
assays. Alternatively, genes encoding two different domains or proteins
can be fused to the wild-type and mutant LexA DBD in the proper frame,
and heterodimerization occurring in SU202 can be quantitated in the
same fashion (Fig. 1). In this study we used pSR658, carrying the
wild-type LexA DBD, and pSR659, expressing the mutant LexA DBD.
View larger version (26K):
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FIG. 1.
Modified LexA-based system for investigating
protein-protein interactions. The LexA-based system was based on that
of Dmitrova et al. (6). A homodimerizing fusion expressed
from one of the wild-type (wt) LexA DBD::MCS plasmids
(pSR658, pSR660, or pSR662) will bind to the wild-type LexA operator
and repress expression of lacZ in the chromosome of the
reporter strain SU101. A heterodimerizing fusion, one subunit expressed
from one of the wild-type LexA DBD::MCS plasmids and the
other from one of the mutated (mut) LexA DBD::MCS plasmids
(pSR659, pSR661, or pSR663), will bind to the hybrid LexA operator and
repress expression of lacZ in the chromosome of the reporter
strain SU202. The choice of plasmid MCS (A, B, or C) is made according
to the reading frame of the desired fusion. Wild-type and mutated
DBD correspond to LexA1-87WT and
LexA1-87408, respectively (6).
Full-length protein fusions can homodimerize.
The four genes
involved in the synthesis and activation of sialic acid in E. coli K1, neuA, neuB, neuC, and
neuD, were fused in frame to the wild-type LexA DBD in
pSR658. To determine that the plasmids were expressing protein when
induced, we performed a Western blot analysis using induced SU101 cells
carrying each plasmid grown to stationary phase in 1 mM IPTG.
Twenty-microliter aliquots of cells were pelleted, resuspended in 15 µl of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) sample buffer with -mercaptoethanol, and incubated at
95°C for 5 min. The samples were resolved by SDS-PAGE on a 12%
polyacrylamide gel. After separation, the proteins were transferred to
a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, Calif.) and
probed with anti-LexA antibody (Invitrogen), using standard procedures
(2). The blot was detected using an enhanced
chemiluminescence detection system (Amersham Pharmacia Biotech,
Piscataway, N.J.). The LexA fusions added approximately 9.6 kDa to the
protein of interest. All full-length neu gene fusions were
expressed, and their mobilities on an SDS-polyacrylamide gel exhibited
the expected pattern (Fig. 2).
|
-(2,8)-polysialic acid capsule (H.46) (22).
Measuring repression of -galactosidase activity in induced cells of
strain SU101 carrying each fusion allowed quantitation of
homodimerization of full-length proteins fused to the wild-type LexA
DBD in pSR658. For these experiments, only those fusion proteins that
repressed expression of -galactosidase at a level that resulted in a
pale colony color of the reporter strain on MacConkey agar plates were
considered to be interacting efficiently. Quantitatively, the amount of
repression required to display a discernibly different colony color was
on average 
25% of the control level. Cells were grown to stationary
phase with the appropriate antibiotics and 1 mM IPTG. Then 100 µl of
each culture was used to inoculate 2 ml of fresh Luria-Bertani medium
with IPTG and antibiotics. The cultures were grown to log phase and
harvested; -Galactosidase activity was determined as described by
Miller (10). The units were calculated from the results of
at least three independent cultures assayed in triplicate. Percent
repression relative to the control cultures was calculated as
[1.0 
(Miller units of sample/Miller units of control)] × 100. The CMP-NeuNAc synthase protein, NeuA, fused to wild-type LexA DBD
repressed -galactosidase activity by an average of 78%, compared to
SU101 with pSR658 alone (Table 1). This
is consistent with the observation that NeuA is enzymatically active as
a dimer (W. F. Vann and D. Stoughton, unpublished data). The NeuC
fusion repressed at 68%, while NeuB, NeuNAc synthase, repressed
expression of -galactosidase by 39%. The fusion with NeuD was the
most efficient of the capsule synthesis proteins, exhibiting 87%
repression. NeuD belongs to a family of acetyl- or acyltransferases
that includes LpxA, LacA, and CysE (1, 7, 18). Like other
members of this family, NeuD contains a signature hexapeptide repeat
motif referred to as an isoleucine patch (7, 18). The
crystal structures of three hexapeptide proteins have been determined;
they each form trimers and share a unique structural feature known as a
left-handed parallel helix (3, 14). NeuD also forms
trimers of identical subunits in vivo (5). These
observations are consistent with the high level of NeuD
homodimerization observed with the LexA system.
|
-galactosidase
expression in this system was confirmed by fusing a gene encoding the
type I chloramphenicol acetyltransferase from Tn9 to the
LexA DBD in pSR658. Chloramphenicol acetyltransferase is a
well-characterized homotrimer and belongs to the same family of acetyl-
or acyltransferases as NeuD. This construct conferred chloramphenicol
resistance to DH5 cells, and its expression was detected via
immunoblot using anti-LexA antiserum (data not shown). The fusion
protein repressed -galactosidase expression in SU101 by 94% (Table
1). The reporter strain SU101 contains a Tn9 insertion in
its chromosomal copy of lacZ and constitutively expresses
unfused type I chloramphenicol acetyltransferase subunits. The high
level of repression that we observed indicates that the modified
LexA-based system is useful in determining interactions between fusion
proteins even in the presence of wild-type subunits expressed by the
host cell.
Fragments of neu genes do not homodimerize efficiently. In contrast to the results with full-length protein fusions, fragments of the neu genes did not yield fusion proteins that homodimerized as efficiently. For these experiments, three fragments of neuD (neuD1 to neuD3) and a fragment (neuA1) encoding the amino-terminal 250 amino acids of neuA (Table 1) were amplified by PCR and fused to pSR658 in frame with the LexA DBD. These plasmids were then used to transform SU101. Since the fragment fusions could not be identified by complementation, the transformants were selected via antibiotic resistance and the inserts were confirmed by colony PCR with primers that flanked the region of interest, by restriction endonuclease digests of purified plasmid preparations, and by DNA sequencing. We were unable to detect the fragment fusions on a Western blot probed with anti-LexA antiserum. The inability to detect small fusions on an immunoblot was also reported by Enz et al. in their study of FecA and FecR fragments replacing the Jun and Fos zipper regions in the parent vectors pDP804 and pMS604 (8). However, the ability of the NeuD2 fragment to heterodimerize with NeuB (see below) indicates that the NeuD2 fragment fusion was expressed. Moreover, the NeuA1, NeuD1, and NeuD3 fusions were positive on a dot blot probed with anti-LexA antiserum (data not shown). While this does not confirm the size of the small fragment fusions, it does establish that they are indeed expressed.
Unlike the full-length NeuA fusion, the NeuA1 fusion did not homodimerize, as reflected by its inability to efficiently repress-galactosidase expression (Table 1). Interestingly, a truncated protein encoding the first 250 amino acids of NeuA displayed
approximately 10% of the enzymatic activity of the full-length enzyme
in vitro and formed only transient dimers (Vann and Stoughton,
unpublished). The NeuD fragment fusions each repressed
-galactosidase expression at a much lower level than full-length
NeuD (Table 1). Fragmentation of NeuD likely resulted in the inability
of the fusions to fold properly, and as a result they homodimerized poorly.
NeuB and NeuD interact in vivo.
The data in Table 1
indicate that the LexA-based system can be used to study
homodimerization of full-length proteins. To investigate the
suitability of the modified LexA system to study the protein-protein
interactions among different components of the kps complex,
we examined the interaction between the NeuD and NeuB proteins. Both
proteins are involved in sialic acid synthesis and are likely to form a
complex in vivo (13). For these experiments, full-length
neuB and neuD genes, as well as fragments of
neuD, were fused in frame to either the wild-type (pSR658)
or mutant (pSR659) lexA DBD and introduced into the reporter
strain SU202. As shown in Table 2, we
observed significant interaction between NeuD and NeuB, quantitated as
71% repression of -galactosidase activity, in cells harboring the
NeuB fusion expressed from the high-copy-number vector, pSR658, and a
NeuD fusion in the lower-copy-number vector, pSR659.
|
-galactosidase expression in SU202 harboring the full-length NeuB fusion in pSR658 and pSR659 carrying fragments of
NeuD. Only one NeuD fragment, NeuD2, displayed interaction approaching that of full-length NeuD with the full-length NeuB fusion.
Fragment NeuD2, when expressed from pSR659, repressed -galactosidase
expression 66%.
These data suggest that NeuD and NeuB interact in vivo and that amino
acid residues 90 to 207 of the NeuD protein are important for this
interaction. However, the formation of heterodimers by two proteins is
not necessarily straightforward when one of the proteins, like NeuD, is
able to form homodimers, and we did observe conflicting results with
the reciprocal experiment. When the neuD gene, fused to the
lexA DBD in pSR658 was introduced into SU202 harboring
neuB fused to the mutant lexA DBD in pSR659,
little repression of -galactosidase activity was seen (Table 2). We postulate that when NeuD is expressed from pSR658, the high-copy-number vector, it is more likely to form homodimers and less likely to interact with NeuB and repress the hybrid promoter in SU202. This view
is supported by the observation that the NeuD2 fragment of NeuD, which,
in contrast to the full-length protein, is unable to efficiently
homodimerize (Table 1), effectively interacted with NeuB even when
expressed from the higher-copy-number vector (Table 2). This was
reflected in 49% repression of -galactosidase activity.
Conclusions.
In this communication we describe the use of a
modified LexA-based genetic system to investigate, in vivo, the
interactions between proteins that comprise a putative
hetero-oligomeric complex responsible for K1 capsule synthesis (4,
13). Both homo- and heterodimerization of protein fusions were
detected. This system allows determination of the heterodimerization of
two fusion proteins even when one protein homodimerizes strongly, as is
the case with NeuD. The system can also reveal the interaction of fusion proteins in the presence of unfused subunits expressed by the
host cell, as shown by the chloramphenicol acetyltransferase experiment. Only those fusion proteins that repressed expression of
-galactosidase at a level that resulted in a pale colony color of
the reporter strain on MacConkey agar plates (25% of the controls) were considered to be interacting efficiently. That is not to say,
however, that repression below 25% may not be biologically significant. However, the limitations of any LexA-based system include
difficulty in deducing weak or transient interactions, steric hindrance
by the fusion moiety, and sequestration of the fused moiety into an
intracellular complex (9). Moreover, the inability to detect
heterodimerization does not preclude association of a given protein
with a higher-order multimer of another protein. It is unlikely that
such interactions would be detected in this system. The different copy
numbers of the vectors, although useful in determining the
heterodimerization of a strongly homodimerizing protein by allowing
reciprocal fusions, add a level of complexity to the system, and it is
recommended that any putative heterodimerizing fusions also be assayed
when expressed from the opposite vector pair. It is also possible that
overexpression of a wild-type LexA fusion that strongly homodimerizes,
may, to some degree, overcome the low affinity of the wild-type LexA
DBD for the hybrid operator in SU202. With these limitations in mind,
we consider this modified system a fast, economical, and reproducible
tool to initially screen for possible interactions and to be suitable
for use in conjunction with biochemical methods for demonstrating
protein-protein interactions, such as gel filtration and protein
cross-linking.
| |
ACKNOWLEDGMENTS |
|---|
We are extremely grateful to M. Granger-Schnarr for her kind gift of plasmids pDP804 and pMS604 and the reporter strains SU101 and SU202. We also thank Virginia Clark, Marty Pavelka, and Lou Passador for critically reviewing the manuscript.
This work was supported by NIH grants AI39615 to R.P.S. D.A.D. was supported by Molecular Pathogenesis of Bacteria and Viruses training grant AI07362 from the Public Health Service.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: University of Rochester Medical Center, Department of Microbiology and Immunology, 601 Elmwood Ave., Box 672, Rochester, NY 14642. Phone: (716) 275-0680. Fax: (716) 473-9573. E-mail: rips{at}uhura.cc.rochester.edu.
Present address: Department of Molecular Microbiology and
Immunology, University of Missouri
Columbia, Columbia, MO 65212.
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Journal of Bacteriology, September 2000, p. 5267-5270, Vol. 182, No. 18
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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