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Journal of Bacteriology, November 1998, p. 5875-5884, Vol. 180, No. 22
Department of Molecular Biology, Princeton
University, Princeton, New Jersey 08544,1
and
Department of Microbiology and Immunology, Uniformed
Services University of the Health Sciences, Bethesda, Maryland
20814-47992
Received 7 July 1998/Accepted 18 September 1998
In Escherichia coli, transcription of the
degP locus, which encodes a heat-shock-inducible
periplasmic protease, is controlled by two parallel signal transduction
systems that each monitor extracytoplasmic protein physiology. For
example, the heat-shock-inducible sigma factor, In Escherichia coli,
transcription of the degP gene, which encodes a
heat-shock-inducible periplasmic protease, is modulated by at least two
signal transduction systems that function in parallel with respect to
each other. The Cpx signal transduction pathway and the
To further characterize the transcriptional regulation of
degP by Cpx and The wec gene cluster is required for the synthesis of the
enterobacterial common antigen (ECA), a glycolipid found in the outer
leaflet of the outer membrane in all species of the family Enterobacteriaceae (reviewed in references 11,
12, and 24). The polysaccharide portion of
ECA consists of three sugar moieties: N-acetyl-D-glucosamine (GlcNAc),
N-acetyl-D-mannosaminuronic acid (ManNAcA), and
4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc). These sugars
form a linear trimeric repeat with the following structure:
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Accumulation of the Enterobacterial Common
Antigen Lipid II Biosynthetic Intermediate Stimulates
degP Transcription in Escherichia
coli
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
E,
controls degP transcription in response to the
overproduction and folded state of various extracytoplasmic proteins.
Similarly, the CpxA/R two-component signal transduction system
increases degP transcription in response to the
overproduction of a variety of extracytoplasmic proteins. Since
degP transcription is attuned to the physiology of
extracytoplasmic proteins, we were interested in identifying negative
transcriptional regulators of degP. To this end, we
screened for null mutations that increased transcription from a strain
containing a degP-lacZ reporter fusion. Through this
approach, we identified null mutations in the wecE,
rmlAECA, and wecF loci that
increase degP transcription. Interestingly, each of these
loci is responsible for synthesis of the enterobacterial common antigen
(ECA), a glycolipid situated on the outer leaflet of the outer membrane
of members of the family Enterobacteriaceae. However, these
null mutations do not stimulate degP transcription by
eliminating ECA biosynthesis. Rather, the wecE,
rmlAECA, and wecF null mutations
each impede the same step in ECA biosynthesis, and it is the
accumulation of the ECA biosynthetic intermediate, lipid II, that
causes the observed perturbations. For example, the lipid
II-accumulating mutant strains each (i) confer upon E. coli
a sensitivity to bile salts, (ii) confer a sensitivity to the synthesis
of the outer membrane protein LamB, and (iii) stimulate both the Cpx
pathway and E activity. These phenotypes suggest that
the accumulation of lipid II perturbs the structure of the bacterial
outer membrane. Furthermore, these results underscore the notion that
although the Cpx and E systems function in parallel to
regulate degP transcription, they can be simultaneously
activated by the same perturbation.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
E regulatory system each control degP
transcription in response to extracytoplasmic signals (4, 6, 15,
19, 21, 22). For example, overproduction of outer membrane
proteins increases E activity, while mutations that
decrease the production of outer membrane proteins concomitantly
decrease E activity. The signal transduction system that
senses these extracytoplasmic changes has recently been defined, and it
consists of an inner membrane anti-sigma factor (RseA) and a
periplasmic protein (RseB) that both monitor extracytoplasmic protein
physiology (7, 18). A second signal transduction system,
comprised of the CpxA-CpxR two-component proteins, also regulates
degP transcription in response to extracytoplasmic protein
physiology (4, 6, 9, 19, 32).
E, we screened for negative
regulators of degP transcription. Through this approach, we
identified null mutations in three genes of the wec gene
cluster that stimulate degP transcription.
3)
-D-Fuc4NAc-(14)-
-D-ManNAcA-(14)--D-GlcNAc-(1) (Fig. 1) (13). Each
polysaccharide chain is ultimately linked to phosphatidic acid via a
phosphodiester linkage.
View larger version (13K):
[in a new window]
FIG. 1.
The trisaccharide repeat unit of the ECA polysaccharide
moiety. This repeat unit,
-D-Fuc4NAc-(14)--D-ManNAcA-(14)--D-GlcNAc,
constitutes the amino sugar polymer of ECA.
N-Acetylglucosamine (GlcNAc) also serves as the attachment
site for the lipid anchor.
N-Acetyl-D-mannosaminuronic acid and
4-acetamido-4,6-dideoxy-D-galactose are abbreviated as
ManNAcA and Fuc4NAc, respectively. The dashed line representing the
bond between the number 6 oxygen and the acetyl group of GlcNAc
indicates that the acetyl group is not present in stoichiometric
amounts in ECA.
The ECA polysaccharide trimer is synthesized in stepwise fashion at the inner membrane by the successive transfer of the component amino sugars to the lipid carrier, undecaprenyl monophosphate (Fig. 2a). The first step in ECA synthesis is the transfer of GlcNAc-1-phosphate from UDP-GlcNAc to undecaprenyl monophosphate (C55-P) to yield C55-PP-GlcNAc, also known as lipid I. ManNAcA is then transferred from UDP-ManNAcA to lipid I to yield C55-PP-GlcNAc-ManNAcA (lipid II). Finally, Fuc4NAc is transferred from TDP-Fuc4NAc to lipid II to yield a complete lipid-linked trimer, C55-PP-GlcNAc-ManNAcA-Fuc4NAc (lipid III) (1, 16, 25). Subsequent steps involve polymerization, transfer of the polymer to a phospholipid aglycone, and translocation to the outer membrane. The nature and chronology of these last three events have not yet been established.
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The mutations identified in this study all interfere with the
conversion of lipid II to lipid III (Fig. 2). Indeed, the results described here indicate that the accumulation of lipid II stimulates degP transcription. Specifically, the lipid II-accumulating
mutants can (i) confer upon E. coli a sensitivity to bile
salts, (ii) confer a sensitivity to the synthesis of the outer membrane
protein LamB, and (iii) stimulate both the Cpx pathway and
E activity. These phenotypes suggest that the
wec mutations perturb the structure of the bacterial outer
membrane. Moreover, analysis of these mutations underscores the notion
that although the Cpx and E systems function in parallel
to regulate degP transcription, they can be simultaneously
activated by the same perturbation (4, 9, 19).
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Media and reagents. Media were prepared as described by Silhavy et al. (28). Liquid cultures were grown in Luria broth. Unless specifically noted, the final concentrations of antibiotics used in growth media were as follows: ampicillin, 50 µg/ml; spectinomycin, 50 µg/ml; and chloramphenicol, 20 µg/ml. Standard microbiological techniques were used for strain construction and bacterial growth (28). 5-Bromo-4-chloro-3-indolyl-D-galactoside (X-Gal) was purchased from Fischer.
Strains and phage.
All strain genotypes are provided in the
text and figure legends. 
RS88, RS45, and NK1324 have been
described elsewhere (10, 29). The degP-lacZ and
rpoHP3-lacZ operon fusions have been
described elsewhere (6, 15).
Screening for negative regulators of degP
transcription.
Nine independent cultures of MC4100 were infected
with NK1324 (which delivers the Tn10cam transposon) as
described by Kleckner et al. (10). Infected cells were then
plated on Luria agar containing chloramphenicol to select for cells
carrying Tn10cam insertions. The resulting transductants
were grouped into nine independent pools, with each pool containing at
least 1,200 colonies.
RS88[degP-lacZ]). Transductants were plated on Luria
agar containing chloramphenicol and 1.4 µg of X-Gal per ml. The
parent strain (PND257) is phenotypically Lac
(white) on
this medium, providing a simple screen for mutants with increased
degP transcription. Eight Lac+ (blue) colonies
were isolated from each of the nine pools and further analyzed.
The Tn10cam insertions within each Lac+ mutant
were reintroduced into the parent strain (PND257) to determine whether
the increase in Lac activity was due to the specific Tn10cam
insertion. Those mutant strains whose increased Lac activity resulted
from the Tn10cam insertion were further analyzed. These
rebuilt strains were used for subsequent analyses. Each insertion
mutation (except insertion 5) was numbered XY. The
X value indicates the pool number (1 to 9) from which the
mutation was isolated, while the Y value indicates the
isolate number (1 to 8) from the given pool. Insertion 5 was isolated
in a preliminary pilot experiment, and as a consequence, it was not
given a designated pool number.
Determination of -galactosidase activity.
Cells were
grown overnight in Luria broth, then subcultured (1:40) into 2 ml of
the same medium, and grown to mid-exponential phase. -Galactosidase
activities were determined by a microtiter plate assay (29).
-Galactosidase activities are expressed as (units/A600) × 103, where
units = micromoles of product formed per minute. Assays were
performed on a minimum of four independent isolates of each strain, and
the results were averaged to obtain the indicated activities. Error
bars indicate the standard deviations. The absence of error bars
indicates that the standard deviation fell below the resolution limit
of the graphing program.
Passive hemagglutination assay for the presence of ECA. Determination of the presence of ECA was performed as described elsewhere (24).
Determination of chromosomal insertion sites of the Tn10cam insertions. The precise sites of Tn10cam insertions were determined in the following manner. Strains carrying the wecF::cam, rmlAECA::cam, and wecE::cam mutations were subjected to arbitrarily primed PCR (2) using the CAM-5' primer (5' CTG ACG GGG TGG TGC GTA ACG GC 3') and the ARB1 primer (5' GG CCA CGC GTC GAC TAG TAC NNN NNN NNN NGA TAT 3'). The PCR products generated by these two primers were subjected to a secondary amplification step with the CAM-5' primer and the ARB2 primer (5' GGC CAC GCG TCG ACT AGT AC 3'). PCR products generated from the second amplification step were sequenced, and the junction between the Tn10cam sequence and the host chromosomal DNA was determined from this sequence information.
Specifically, the Tn10cam of mutant 5 (wecF::cam) is inserted between nucleotides 7863 and 7864 of the published wec gene cluster sequence (accession no. AE000455). The Tn10cam of mutant 31 (wecE::cam) is inserted between nucleotides 5707 and 5708 of the published wec gene cluster sequence, while the Tn10cam of insertion 22 (rmlAECA::cam) is situated between nucleotides 3894 and 3895 of the same sequence.Assay for lipid II accumulation. The incorporation of [3H]GlcNAc into lipid II was determined as previously described (26). Briefly, bacteria were grown with vigorous aeration at 37°C in 60 ml of Proteose Peptone-beef extract medium supplemented with glucose (0.2% final concentration) to an A600 of 0.4. The cells were then harvested by centrifugation, resuspended in fresh Proteose Peptone-beef extract-0.2% glucose (6 ml), and incubated at 37°C with [3H]GlcNAc (75 µCi, 5.2 Ci/mmol) for 30 min. The labeled cells were subsequently poured over crushed ice, harvested by centrifugation, and washed with cold 0.9% saline. The washed cells were then successively extracted with 95% ethanol (6 ml) and acetone (6 ml) and then dried in vacuo. The dried cells were extracted with chloroform-methanol (3:2 [vol/vol]), and the extracts were analyzed by ascending paper chromatography on EDTA-treated silica-gel-impregnated paper with chloroform-methanol-water-concentrated ammonium hydroxide (88:48:10:1 [vol/vol/vol/vol]) as the developing solvent. The material in the region of the chromatogram corresponding to lipid II was eluted, and the amount of radioactivity in this material was determined. In addition, the identity of this material as lipid II disaccharide was verified by treatment of the material with mild acid followed by gel filtration chromatography of the water-soluble fraction on a Bio-Gel P2 column.
Maltose sensitivity disc assays. The maltose sensitivity disc assays, whose results are shown in Fig. 6, were performed as follows. Each strain was grown to saturation overnight at 37°C in 5 ml of Luria broth. Three milliliters of molten Luria top agar (55°C) was mixed with 100 µl of an overnight culture and immediately spread onto Luria agar (warmed to 23°C). The top agar was allowed to solidify for 2 min. A Schleicher and Schuell analytical paper filter disc (7-mm diameter) was then placed in the middle of the Luria agar plate. Ten microliters of 20% (wt/vol) maltose was placed on the filter disc, and the plates were incubated overnight at 37°C. The zone of clearing, which is defined as the diameter of inhibited growth minus the diameter of the filter disc, was measured 18 h after the inception of incubation. Each value shown in Fig. 6 is the average of four replicate experiments. The error bars represent the standard deviations from each average.
Genetic nomenclature. Genes and gene products involved in ECA and O-antigen synthesis and assembly are designated in accordance with the recently formulated bacterial polysaccharide gene nomenclature scheme (23). The following are the relevant new designations, each of which is accompanied by the former designation in parentheses: wbb (rfb), wec (rfe/rff), wecA (rfe), wzzECA? (o349), wecB (rffE), wecC (rffD), rmlB (o355), rmlA (o292), wecD (rffC), wecE (rffA), wzxECA? (o416), wecF (rffT), and wecG (rffM). In addition, the subscripts ECA and Oag have been included where appropriate to distinguish between homologs involved in ECA and O-antigen synthesis, respectively.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Rationale and mutant isolation.
We sought to identify negative
regulators of degP transcription by generating null
mutations that increased transcription from a degP-lacZ
reporter fusion. To facilitate this analysis, we created strain PND257
(MC4100; ompR::Tn10
RS88[degP-lacZ]), which contains the
degP-lacZ fusion as well as the
ompR::Tn10 null mutation.
ompR null strains do not synthesize the major outer membrane
proteins OmpC and OmpF (30), and consequently,
E activity is reduced in such strains (15).
Because of the reduction in E activity, PND257 is
phenotypically Lac (white) on Luria agar containing 1.4 µg of X-Gal per ml. This Lac phenotype provides a
simple screen for mutants with increased degP transcription.
By generating null mutations throughout the chromosome of PND257, we
hoped to identify mutants with increased Lac activity, and by
extension, mutations that impaired the function of negative regulators
of degP transcription.
NK1324, which delivers the Tn10cam transposon
(10), to perform transposon mutagenesis on strain PND257.
The chloramphenicol-resistant colonies generated by this procedure were
screened for those with increased degP transcription.
Approximately 15,000 chloramphenicol-resistant colonies were screened,
and 11 colonies with increased degP transcription were
analyzed. Of these 11 isolates, 9 grew poorly on lactose-MacConkey agar, while the remaining two grew as well as the parent (PND257) on
this medium. Since sensitivity to MacConkey agar is often an indicator
of a structural perturbation in the outer membrane (20), and
since degP transcription is attuned to such perturbations (15, 22, 27), we chose to analyze the nine MacConkey
agar-sensitive mutants.
Figure 3 shows that each
transposon-generated insertion increases degP transcription
approximately three- to sixfold compared to the parent strain. For
example, insertions 5 and 31 increase degP transcription
approximately sixfold (Fig. 3, compare lanes 2 and 4 with lane 1),
while insertion 22 increases degP transcription approximately threefold (Fig. 3, compare lanes 1 and 3). The six remaining transposon-generated mutations increase degP
transcription to the same extent as that observed with insertions 5 and
31 (data not shown).
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Null mutations within the wec gene cluster stimulate
degP transcription.
The transposon insertions in each
of the MacConkey agar-sensitive isolates were all tightly linked to the
wec gene cluster, which is located at approximately 85 min
on the E. coli chromosome. Since the wec gene
cluster is involved in the biosynthesis of ECA, we were interested in
determining whether the Tn10cam insertions affected the
biosynthesis of ECA. Using a passive hemagglutination assay for ECA
biosynthesis (25), we determined that eight of the nine
insertions did not produce ECA (ECA). Only one insertion
(no. 22) remained ECA+. Interestingly, insertion 22 causes
the weakest induction of degP transcription (Fig. 3).
The rmlAECA::cam
mutation.
The
rmlAECA::cam mutation was
anomalous in two major regards. First, unlike the other eight
mutations, the rmlAECA::cam
mutation did not confer an ECA phenotype, although it
mapped within the wec gene cluster. Second, the
rmlAECA::cam mutation
conferred a relatively weak increase in degP transcription
(approximately 50% of that observed with the other Tn10cam insertions).
phenotype and (ii) display an attenuated increase in
degP transcription compared with the wecF and
wecE mutations, which completely abolish the synthesis of
lipid III. These predictions are borne out in the data described above
(Fig. 3).
One additional prediction is that all of the Tn10cam
insertions should impede the conversion of lipid II to lipid III (Fig. 2). Indeed, analysis of the PND788 (PND257;
wecF::cam), PND789 (rmlAECA::cam), and PND790
(wecE::cam) strains shows that they accumulate significant amounts of lipid II, while their parent strain,
PND257, does not accumulate lipid II in detectable quantities (Table
1). This information indicates that (i)
the mutations in wecF and wecE are
loss-of-function mutations and (ii) this study provides the first
evidence indicating that the rmlAECA gene
product is actually involved in ECA biosynthesis.
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The accumulation of the lipid II ECA biosynthetic intermediate increases degP transcription and confers sensitivity to bile salts. From the data presented, it is clear that all of the mutations identified in our screen affect the same general step in the biosynthesis of ECA. Specifically, the insertions within wecF and wecE cause the accumulation of lipid II while the rmlAECA::cam mutation impedes the conversion of lipid II to lipid III (Fig. 2a).
What is not clear, however, is whether it is the absence of ECA or the accumulation of the lipid II intermediate that stimulates degP transcription and confers sensitivity to MacConkey agar. Various tests of epistasis were performed to distinguish between these two possibilities. For example, the wecA::Tn10 mutation was introduced into the wecF::cam, wecE::cam, and rmlAECA::cam strains, and the resulting amounts of degP transcription were determined (Fig. 4). The wecA::Tn10 mutation blocks ECA synthesis at its earliest step, preventing the transfer of GlcNAc phosphate from UDP-GlcNAc to undecaprenyl monophosphate (Fig. 2a). If the lack of ECA is the cause of the observed phenotypes, the wecA::Tn10 wecE::cam double mutant strain should display at least the same degree of degP transcriptional induction and MacConkey agar sensitivity as observed with the wecE::cam mutation alone. If the accumulation of lipid II is responsible for the transcriptional induction of degP, then the double mutant strain should not display the transcriptional induction observed with the single chloramphenicol-resistant insertion mutations. These predictions also hold true for the wecF::cam and rmlAECA::cam mutant strains.
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phenotype. If this model is correct,
introduction of the wecE::Tn10 mutation
into a strain that contains the
rmlAECA::cam insertion should raise degP transcription to the level observed with
the wecE mutation alone. Again, this prediction is verified.
Specifically, the rmlAECA wecE double
mutant strain displays the same degree of transcriptional induction of
degP as the wecE mutation does in isolation
(compare lanes 2, 4, and 6 in Fig. 4).
There are two general conclusions that can be drawn from Fig. 4. First,
the accumulation of lipid II stimulates degP transcription and confers MacConkey agar sensitivity in these strains. Second, the
rmlAECA::cam mutation
impedes, but does not abolish, the conversion of lipid II to lipid III
in the biosynthesis of ECA.
The induction of degP transcription by accumulation of the lipid II intermediate is decreased in an ompR+ background. Since the experiments described above have all utilized strains that were ompR null, we were also interested in determining the effects of lipid II accumulation in an ompR+ background. Figure 5 shows that while the wec mutations still stimulate degP transcription in the ompR+ background, the magnitude of the induction of degP transcription conferred by these mutations is significantly reduced compared to the analogous ompR strains. These ompR+, lipid II-accumulating strains are also no longer as sensitive to growth on MacConkey agar as their ompR counterparts.
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Accumulation of the lipid II intermediate can confer a sensitivity to high-level synthesis of the outer membrane protein LamB. During the course of this study, we noted that the lipid II-accumulating mutant strains were exquisitely sensitive to growth on maltose and maltodextrins. As a consequence, we wanted to determine if these strains had difficulty growing in the presence of all types of sugars or whether their sensitivity was restricted to growth in the presence of maltose and its oligomers. To this end, we assayed the growth of the lipid II-accumulating strains on Luria agar in the presence of high concentrations of maltose, lactose, and glucose. The lipid II-accumulating strains were sensitive only to maltose, indicating that this sensitivity is not simply a sugar-mediated effect (data not shown).
Since the various phenotypes described for these strains appear to be associated with perturbations in the outer membrane, the observed maltose sensitivity might be due to high-level synthesis and export of the outer membrane porin LamB (LamB synthesis is induced in the presence of maltose). To address this issue, the lamB
60
mutation, which deletes a portion of the LamB signal sequence (and
prevents its export across the inner membrane [8]),
was introduced into the lipid II-accumulating strains. We then
determined if these lamB60 strains were also sensitive to
high levels of maltose. Figure 6
indicates that the lamB60 mutation abolishes the
sensitivity of the lipid II-accumulating strains to high concentrations of maltose (compare lanes 2 and 6, 3 and 7, and 4 and 8). Thus, the
maltose sensitivity observed with these strains is due to high-level
export of wild-type LamB, further suggesting that the accumulation of
lipid II perturbs the physiology of the outer membrane.
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Accumulation of lipid II does not interfere with incorporation or
assembly of LamB in the outer membrane.
Because of the observed
toxicity associated with high-level synthesis of LamB in the lipid
II-accumulating mutants, we wanted to determine if these strains
displayed defects in the incorporation and assembly of LamB into the
outer membrane. To address this issue, we performed two experiments.
(i) We examined the amount of LamB protein associated with membrane
fractions of a parental strain, PND257 (MC4100;
ompR::Tn10
RS88[degP-lacZ]), as well as derivative
strains that accumulate lipid II, PND788 (PND257; wecF::cam), PND789 (PND257;
rmlAECA::cam), and
PND790 (PND257; wecE::cam). (ii) We
also examined the kinetics of LamB trimerization in PND257, PND788,
PND789, and PND790. None of the lipid II-accumulating strains displayed
defects in the incorporation of LamB into membrane fractions, nor did
these mutants display defects in the trimerization of LamB (data not
shown). Thus, the toxicity associated with high-level synthesis of LamB
in lipid II-accumulating mutants is not the result of a gross
structural defect in LamB assembly.
The wecF::cam,
wecE::cam, and
rmlAECA::cam mutations
increase E activity.
Finally, we were interested in
the mechanism(s) by which degP transcription was being
stimulated as a result of lipid II accumulation. Two regulatory
pathways are known to modulate degP transcription. As
mentioned elsewhere, degP transcription can be increased by increasing E activity, through the RseA/B signal
transduction system (15). In addition, degP
transcription is also regulated by the Cpx two-component signal
transduction pathway (6, 22), which functions in parallel with the E signal transduction pathway (4,
6). To determine the specific route by which the
wecF::cam,
rmlAECA::cam, and
wecE::cam mutations functioned to
increase degP transcription, we introduced each insertion
into SP245 (MC4100; ompR::Tn10
rpoHP3-lacZ). The
rpoHP3 promoter is recognized solely by RNA
polymerases containing the E subunit. Hence, this fusion
provides an assay for only E activity (6,
15). Figure 7 shows that
rpoHP3-lacZ transcription is induced
by the Tn10cam insertions in a qualitatively similar fashion
as that observed with the induction of degP transcription. For example, the wecF::cam and
wecE::cam insertions stimulate rpoHP3-lacZ transcription
approximately 2.6-fold (Fig. 7, compare lane 1 with lanes 2 and 4). The
rmlAECA::cam insertion
stimulates rpoHP3-mediated transcription
approximately 2.3-fold (compare lanes 1 and 3 of Fig. 7).
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The wecF, wecE, and
rmlAECA mutations activate the Cpx signal
transduction pathway.
The results presented in Fig. 7 demonstrate
that the wecF, rmlAECA, and
wecE mutations activate degP transcription, at
least in part by stimulating E activity. However, we
were also interested in determining if these mutations stimulated
degP transcription via the Cpx pathway as well. Accordingly,
the cpxR null mutation was introduced into the parent strain
(PND257) as well as the wecF,
rmlAECA, and wecE mutant strains
(PND788, PND789, and PND790, respectively), and the amount of
degP-lacZ transcription generated from these strains was
quantified. Interestingly, elimination of the Cpx pathway by a
cpxR null mutation decreases, but does not abolish, the
transcriptional induction of degP conferred by the
wecF, rmlAECA, and wecE
mutations (Fig. 8). For example, in a
cpxR background, the
wecF::cam and wecE::cam mutations stimulate
degP transcription only 1.6- to 1.7-fold (Fig. 8, compare
lane 5 with lanes 6 and 8). Similarly, the
rmlAECA::cam mutation
stimulates degP transcription approximately 1.4-fold in the
cpxR background (Fig. 8, compare lanes 5 and 7). Although
the transcriptional induction of degP is qualitatively similar in the cpxR+ and cpxR
background, CpxR is clearly responsible for the majority of the
degP transcriptional induction in the parental background (PND257). For instance, the wecF::cam
mutation stimulates degP transcription approximately 6-fold
in the cpxR+ background, but only 1.7-fold in
the cpxR background (Fig. 8, compare lanes 1 and 2 with
lanes 5 and 6).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In our search for negative regulators of degP
transcription, we have shown that mutations that cause the accumulation
of the lipid II intermediate in the pathway for ECA biosynthesis cause several envelope-associated perturbations. First, these mutations confer a sensitivity to growth in the presence of bile salt detergents, which is a classic indicator of an outer membrane permeability defect
(20). Second, these mutations confer a sensitivity to high-level export of the wild-type LamB porin, also suggesting a
perturbation in outer membrane physiology. Third, these mutations increase degP transcription by stimulating both the
E modulatory signal transduction system (15)
and the Cpx signal transduction system (6, 22). Since
degP encodes a periplasmic protease that destroys misfolded
extracytoplasmic proteins (see reference 17), this
is also an indicator of a perturbation in the physiology of periplasmic
and/or outer membrane proteins. Taken together, these results imply
that lipid II accumulation perturbs the physiology of envelope
proteins, thus causing an increase in degP transcription.
Accumulation of lipid II. The results presented in Fig. 4 demonstrate that it is the accumulation of lipid II that stimulates degP transcription. Moreover, previous studies have also noted a toxicity associated with the accumulation of lipid II. For example, Rick et al. (26) have observed that Salmonella typhimurium strains containing a lesion in rmlAOag (which encodes a homolog of RmlAECA) accumulate the lipid II intermediate and display sensitivity to sodium dodecyl sulfate (SDS). This SDS-sensitive phenotype can be suppressed by mutations that block the accumulation of lipid II.
Despite the body of evidence indicating that lipid II accumulation confers a host of envelope-associated defects, it is unclear why lipid II accumulation exerts these effects. While ECA is not essential for the viability of E. coli, the undecaprenyl carrier lipid that is used to synthesize the ECA trisaccharide is essential. We considered the possibility that lipid II accumulation indirectly impeded the synthesis of the peptidoglycan layer and/or lipopolysaccharide by sequestering undecaprenyl monophosphate (C55-P). However, overproduction of the BacA protein, which is believed to increase the pool of free C55-P (3), had no ameliorative effect on the lipid II-associated phenotypes (6a). Thus, it seems unlikely that the accumulation of lipid II exerts its effects by sequestering C55-P. An alternative model posits that the partially completed ECA trisaccharide may interfere with the biogenesis of envelope proteins, thus altering the permeability of the outer membrane and signaling for increased levels of the envelope protease, DegP. This interference would most likely occur at or near the inner membrane since the lipid II disaccharide remains attached to its undecaprenyl carrier lipid in the inner membrane. According to this view, the accumulation of lipid II in the inner membrane affects some process(es) that is important for outer membrane biogenesis.The involvement of rmlAECA in ECA biosynthesis. The results presented here also represent the first mutational analysis of rmlAECA. Previous studies that have sought mutations in genes involved in ECA biosynthesis have uncovered several loci in the wec and wbb gene clusters. For example, mutations in wecB, wecC, wecD, wecE, wecF, and wecG have all been identified because of their abolition of ECA biosynthesis (Fig. 2) (16).
Indeed, rmlAOag mutations (Fig. 2a) have also been shown to radically reduce (but not abolish) ECA biosynthesis in S. typhimurium (26). However, despite the homology between RmlAECA and RmlAOag, no mutations were ever identified in rmlAECA. Based on the previous analyses and on the results presented here, we suggest that RmlAECA and RmlAOag perform redundant functions for the biosynthesis of ECA. The reasons for suggesting this are threefold. First, RmlAECA and RmlAOag have 65% amino acid sequence identity, and they each display glucose-1-phosphate thymidylyltransferase activity (14). Second, rmlAOag null strains do not completely abolish ECA biosynthesis in S. typhimurium. Finally, Fig. 4 clearly demonstrates that the accumulation of lipid II stimulates degP transcription. If RmlAECA partially contributes to the conversion of lipid II to lipid III, then inactivation of this locus should display an attenuated increase in degP transcription and should remain ECA+. Moreover, when the rmlAECA null mutation is combined with a second mutation that completely abolishes the conversion of lipid II to lipid III (i.e., wecE), the second mutation should raise the transcriptional induction of degP from the attenuated response observed with only the rmlAECA mutation to the strong induction observed with the wecE mutation. All of these predictions are verified by the results presented here. Thus, although the rmlAECA locus appears to be involved in the biosynthesis of ECA, its identification in this role has been lacking because mutational inactivation of this locus is phenotypically masked by its functional homolog, rmlAOag.The lack of porin enhances the susceptibility of E. coli to lipid II accumulation. From the results of Fig. 3 and 5, it is clear that strains lacking the outer membrane porins OmpF and OmpC are more susceptible to the toxic effects of lipid II accumulation. The reasons for this enhanced susceptibility are at present unclear. It is possible that the lack of OmpF and OmpC directly alters the structure of the outer membrane, perhaps making this membrane more susceptible to the perturbations caused by the accumulation of lipid II. For example, lipid II accumulation may increase the ability of the outer membrane to be solubilized by bile salts when OmpF and OmpC are absent.
Alternatively, we note that the lack of porins in an ompR background decreases the expression of theE regulon
(including degP) by approximately fourfold (15).
Since E regulation is involved in responding to
extracytoplasmic protein stresses, ompR strains (i.e.,
E attenuated) may not be equipped to properly cope with
large-scale perturbations, such as those caused by the accumulation of
lipid II. However, since the precise biochemical effect(s) of lipid II
accumulation is not known, we cannot at present distinguish among these possibilities.
The export-associated toxicity of LamB.
To our knowledge, this
study describes the first instance in which export of the wild-type
LamB protein is toxic. The requirement for export of LamB is specific,
as expression of the nonexportable LamB60 mutant is not toxic.
Interestingly, this toxicity is not due to a gross structural defect in
the assembly of LamB. However, we cannot exclude the possibility that a
minor structural alteration in LamB assembly (not detectable by
membrane fractionation or trimerization studies) confers this toxicity.
Alternatively, the transit of large amounts of LamB protein en route to
the outer membrane may confer the toxicity observed in the lipid
II-accumulating strains. Suppressor analysis may ultimately be
informative as to the precise molecular nature of this toxicity.
Transcriptional induction of degP.
Finally, we note that
the lipid II-mediated induction of degP transcription is
mediated by increases in both Cpx and E activity (Fig. 7
and 8).
E are affected by the same types of
extracytoplasmic stresses.
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ACKNOWLEDGMENTS |
|---|
We thank Susan DiRenzo for manuscript preparation and members of the Silhavy laboratory (especially Scott Hande) for comments and suggestions throughout the course of this work.
P.N.D. gratefully acknowledges support from a National Institutes of Health (NIH) training grant (GM07388). This work was supported by an NIGMS grant to T.J.S. (GM34821) and to P.D.R. (GM52882).
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FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544. Phone: (609) 258-5899. Fax: (609) 258-2957. E-mail: tsilhavy{at}molbio.princeton.edu.
Present address: Department of Molecular and Cellular Biology,
Harvard University, Cambridge, MA 02138.
Present address: University of Texas Southwestern Medical School,
Dallas, TX 75235.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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