From the Departement de Biochimie Médicale,
Centre Médical Universitaire, Université de Genève, 1 Rue Michel Servet, 1211 Geneva 4, Switzerland, and the
§ Department of Microbiology, Immunology, and Molecular
Genetics, UCLA, Los Angeles, California 90095
Received for publication, January 17, 2001, and in revised form, March 20, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Escherichia coli responds to
the accumulation of misfolded proteins by inducing the transcription of
heat shock genes. E Heat shock and other environmental stresses result in the
misfolding of polypeptides in all cells. Escherichia coli
responds to the accumulation of misfolded polypeptides by activating
the transcription of heat shock genes. Heat is a drastic stress that leads to protein unfolding in general and triggers two heat shock responses controlled by two distinct RNA polymerase species in E. coli1:
Previous work identified several genes that are transcribed by
E Earlier work described the isolation of rpoE knockout
mutations (16, 17). E. coli appears to require
rpoE for viability and growth under physiological
conditions, as the mutant strains cope with loss of rpoE
function by acquiring compensatory mutations (18). The nature of
compensatory mutations as well as the number and identity of the
affected genes are still unknown. Even though rpoE seems to
be essential, none of the known E Bacterial Strains and Growth Conditions--
Most strains used
in this study are listed in Table I.
Strains carrying promoter fusions of hitherto unknown genes are
referred to as ecf-lacZ. Sequences of primers used in this
study can be obtained from the authors upon request. Luria Bertani
(LB), MacConkey, and M9 minimal media were prepared as described (19).
When necessary, media were supplemented with 100 µg/ml ampicillin, 50 µg/ml spectinomycin, 15 µg/ml tetracycline, 50 µg/ml kanamycin,
or 20 µg/ml chloramphenicol. The indicator dye
5-bromo-4-chloro-3-indolyl- Construction of Promoter Fusion Librairies--
A library of
chromosomal transcriptional fusions was constructed using
Cloning Procedures and Gene Replacement--
The DNA regions
corresponding to E Primer Extension Analysis--
Total RNA was isolated using the
RNeasy kit from Qiagen. Cultures were grown at 30 °C, and aliquots
were shifted to 50 °C for a period of either 5 or 10 min.
Immediately after the heat shock, cultures were lysed with guanidinium
isothiocyanate following the protocol of the RNeasy kit. To define the
transcriptional start site(s) of each gene, ~10 ng of complementary
oligonucleotide probe was annealed with 10 µg of total RNA. Strand
extension from the annealed primer was achieved using the avian
myeloblastosis virus reverse transcriptase. Primer extension products
were separated on 8 M urea-containing gels, and their
migration profile was compared by running on the same gel the dideoxy
sequencing reactions using the same oligonucleotide.
Biochemical Assays--
Approximating the Number of E Transposon Mutagenesis to Search for Promoter Fusions to Search for E Characterization of E Some Members of the Some
To test this assumption, ecf gene sequences were disrupted
by insertion of
Cointegrates that formed after plasmid insertion into ecfE
and ecfL could not be resolved, suggesting that these genes
may be essential for viability and growth of E. coli. This
hypothesis was tested by repeating the resolution of cointegrate
strains after transformation with a second plasmid, containing the
wild-type ecf gene under control of the arabinose-inducible
araBAD promoter. Streaking cointegrates on
arabinose-containing sucrose plates produced the desired knockout
mutants, whereas streaking on sucrose media without arabinose failed to
produce any colonies. Thus, the chromosomal copy of E. coli
expressing plasmid-encoded ecfE or ecfL can be
deleted by homologous recombination. The resulting strains henceforth
require arabinose-containing media for growth, indicating that
ecfE and ecfL are essential genes.
Synthetic Lethality and Synthetic Conditional Phenotypes for
Cold Shock Weakly Induces the E Two Two new members of the RpoE regulon were observed to be
essential: ecfE and ecfL. Because these genes
appear to be transcribed by several RNA polymerases (data not shown)
and have no definitive function attributed, it is impossible to draw
conclusions as to why the In summary, the E RNA polymerase controls one of the
two heat shock regulons of E. coli. This regulon is
activated upon accumulation of misfolded polypeptides in the double
membrane envelope of E. coli.
E (RpoE) is a
member of the extracytoplasmic function subfamily of sigma factors.
Here we asked how many genes are activated by E
E RNA
polymerase and what is the identity of these genes. Using two
independent genetic approaches, 20 E. coli promoters were identified which activate reporter gene transcription in a
E-dependent manner. In all cases examined, a
canonical
E binding site could be revealed upon mapping
transcriptional start sites. 10 identified promoters activated the
transcription of previously identified genes with four genes acting
directly on the folding of E. coli envelope proteins
(dsbC, fkpA, skp, and surA). The remaining promoters transcribed genes that are
presumed to encode hitherto unknown extracytoplasmic functions and were named ecf (ecfA-ecfM). Two of
these ecf genes were found to be essential for E. coli growth.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
'
32 and
2
'
E, E
32, and
E
E, respectively (1, 2). The unfolding of proteins in
the envelope of E. coli uniquely induces the
E regulon but not E
32 (3, 4).
E (RpoE) is a member of the extracytoplasmic function
(ECF) subfamily of sigma factors which function as effector molecules
responding to extracytoplasmic stimuli (3, 5). Some microorganisms such
as Streptomyces coelicolor harbor multiple ECFs that seem specialized in responding to different extracytoplasmic stimuli (5, 6).
The E. coli
E regulon is induced specifically
in response to imbalanced synthesis of outer membrane proteins (7) and
to misfolding of polypeptides that have been translocated across the
cytoplasmic membrane (8).
E (4). E
E directs its own expression.
rpoE is the first gene of an operon that also contains
rseA, rseB, and rseC
(regulator of sigma E, genes A, B, and
C (9, 10). RseA is a short hydrophobic polypeptide that integrates into
the cytoplasmic membrane. The N-terminal cytoplasmic domain of RseA
binds to
E, sequestering the sigma factor from core RNA
polymerase (E) (9, 10). The C-terminal domain of RseA protrudes into
the periplasm, a compartment located between the cytoplasmic and outer
membranes of E. coli. The C-terminal domain of RseA
interacts with RseB, a periplasmic soluble protein (9, 10). RseB is
believed to sense the concentration of misfolded
polypeptides, causing RseB dissociation from RseA and
liberating cytoplasmic
E for interaction with core RNA
polymerase (11). Another model suggested proteolytic cleavage of RseA
in response to the accumulation of outer membrane proteins (12). The
function of RseC, encoded by the fourth gene of the rpoE
operon, remains unknown. E
E also transcribes
htrA and fkpA, encoding a periplasmic protease (HtrA/DegP) for the removal of misfolded polypeptides (13, 14) and a
periplasmic peptidyl prolyl isomerase (FkpA) involved in folding
envelope proteins (8, 15). rpoH, encoding the transcription factor
32 for the cytoplasmic heat shock response, is
also transcribed by E
E (14).
E-transcribed genes
(rpoH, htrA, fkpA, rseA,
rseB, rseC) is required for either growth or
viability of E. coli. Taken together, all previous work
suggests that E
E must transcribe additional genes that
are involved in the folding of envelope proteins. To identify genes
that are transcribed by E
E and to approximate the size
of the
E regulon, we have used two different
experimental strategies. Small DNA segments, generated by fragmentation
of the E. coli chromosome, were fused to a promoterless
lacZ reporter gene carried on a single copy plasmid.
Further, the
Mu53-lacZ transposon was used to generate
sets of random fusion between the promoterless lacZ reporter
and regulatory sequences of the chromosome of E. coli.
Screening of both libraries of reporter fusions in various genetic
backgrounds identified 20 promoters that activated LacZ expression in a
E-dependent manner. A hypothesis is
presented to account for the essential function of the
E
regulon and to describe the role of the identified genes in responding to misfolded polypeptides within the envelope of E. coli.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside was used
at a final concentration of 40 µg/ml in the agar medium. Mutations
were transduced into various backgrounds using P1 bacteriophage (19).
Labeling experiments using [35S]methionine in the M9 high
sulfur medium were performed as described previously (20).
Bacterial strains
Mu53-lacZ (KanR) (21). Briefly, strain MC4100
(LacZ
) was mutagenized at 30 °C with
Mu53-lacZ, and colonies were formed on MacConkey plates
at 30 °C and 43 °C. Colonies that developed red staining at
43 °C but not at 30 °C were isolated. The site of
Mu53
insertions into the chromosome was determined by DNA sequencing with
the oligonucleotide primer (5'-GTCATAGCTGTTTCCTGTGTG-3'). For this
step, DNA regions carrying the
Mu53-lacZ fusions were cloned into a cosmid, taking advantage of the
Mu53-lacZ
(KanR) marker. A second library was constructed
using the single copy F-based promoter probe vector pFZY (22)
essentially as described earlier (23). Putative
E
E-regulated promoters identified with this strategy
were analyzed by DNA sequencing using the synthetic oligonucleotide
described above.
E-regulated promoters were amplified
by polymerase chain reaction using appropriate primers and cloned into
pRS550 using restriction sites BamHI and EcoRI (24). Plasmids were characterized by DNA sequence analysis, and each
fusion was transferred on the chromosome of strain MC4100 using
bacteriophage
RS45 (24). Null alleles of the newly identified ecf genes were obtained by cloning wild-type ecf
genes into the low copy number plasmid pWKS30 (25). The genes were
disrupted with cassettes carrying resistance to either tetracycline or
kanamycin (26). The disrupted genes were cloned into the unique
SmaI site of pKO3 which carries a temperature-sensitive
replicon as well as the sacB gene for counterselection (27).
Recombinants were transformed into strain MC4100 at 42 °C to select
for cointegrate formation, and they were subsequently streaked at
30 °C on LB-Tet or LB-Kan plates supplemented with 5% sucrose.
Strains that had lost the plasmid (loss of CmR) but
retained the resistance marker of the knockout allele were analyzed
further. Failure to lose the CmR resistance provided by the
suicide plasmid was indicative of merodiploidy, which was observed for
ecfA, ecfE, and ecfL. Each of the
three genes was placed under the arabinose-inducible promoter of pBAD-A
vector (Invitrogen) and expressed in LMG194 background. ecfE::
Tet and ecfL::
Kan
could be recombined on the chromosome from plasmid pKO3 only when the
cells were grown in the presence of arabinose (0.2%). The presence of
the ecfE::
Tet or
ecfL::
Kan alleles in the merodiploid cells
(LMG194/pBAD-ecfE+ and
LMG194/pBAD-ecfL+, respectively) was verified by
transducing linked markers with bacteriophage P1. In all cases,
disruption of ecf genes was verified by polymerase chain
reaction amplification using chromosomal template DNA and appropriate primers.
-Galactosidase activity was
determined as described previously (19). Bacterial cultures were grown
overnight at 30 °C, diluted 1:100, and allowed to reach
A595 nm between 0.5 and 0.7. Aliquots
were maintained or shifted to 14, 37, or 43 °C for 20 min.
Measurements were performed in duplicate, and the data represent the average of at least three independent experiments. Two-dimensional equilibrium gel electrophoresis was performed as described previously (28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E-regulated
Genes--
To measure the size of the RpoE regulon, we analyzed
pulse-labeled E. coli proteins by two-dimensional gel
electrophoresis. This technique has been employed routinely for the
analysis of heat shock regulation (29). E. coli strain
BL21(DE3), carrying a plasmid overexpressing rpoE or an
empty vector control, was grown in M9 minimal medium to
A595 nm 0.6. Cells were pulse labeled with
[35S]methionine for 2 min, and all further incorporation
of radioactivity into polypeptide was quenched by the addition of
excess unlabeled methionine. E. coli cells were lysed in
buffer containing detergent and ampholytes. Proteins in the extracts
were separated by charge electrophoresis of the ampholytes within the
pH range 3.5-10 and then separated on SDS-polyacrylamide gel
electrophoresis in the second dimension based on their molecular mass.
Of the 4,500 polypeptides encoded by the E. coli genome, the
two-dimensional gel electrophoresis technique can identify ~2,000
proteins. Detergent-insoluble, basic, and membrane proteins as well as
polypeptides expressed at very low levels are excluded from the
analysis. To facilitate analysis of the many data spots, black
arrows are positioned in the two panels of Fig.
1 which identify proteins of equal
abundance for both labeling experiments. Compared with the
proteome of wild-type E. coli,
E-overproducing cells contained 13 polypeptide spots
with significantly increased intensity (empty arrows in Fig.
1B), suggesting that these polypeptides represent
E-regulated genes. Further, overexpression of
E appears to have a negative effect on the regulation of
some E. coli genes because the abundance of nine
polypeptides was severely diminished compared with Fig. 1B
(circled peptide spots).
View larger version (105K):
[in a new window]
Fig. 1.
Global effect of rpoE
overexpression as visualized using two-dimensional equilibrium
gel electrophoresis. E. coli BL21(DE3) cultures were
grown at 30 °C to A595 nm 0.6 in M9 medium
supplemented with glucose, and the expression of T7 polymerase was
induced by the addition of 0.5 mM
isopropyl-1-thio- -D-galactopyranoside. After a 30-min
incubation period, cells were labeled with
[35S]methionine for 1 min. Cells were lysed and extracted
with detergents (2% Nonidet P-40) and 8 M urea followed by
several freeze-thawing cycles, and insoluble material was removed by
centrifugation (13,000 × g, 20 min).
35S-Labeled extract supernatant was separated by charge
electrophoresis using a mixture of ampholytes (1.6 and 0.4% in the pH
ranges 5.0-7.0 and 3.5-10.0, respectively) in the first dimension and
a 12.5% SDS-polyacrylamide gel in the second dimension. Autoradiograms
of the two-dimensional gels correspond to extracts of BL21(DE3)
carrying the pEAD vector alone (panel A) or pDM1055
(overexpressing rpoE) (panel B). The
circles in panel A identify proteins of lesser
abundance when
E is overproduced (for comparison, see
panel B). Arrows in panel B identify
proteins with increased abundance when
E is
overproduced. The letters K, EL, and E
identify DnaK, GroEL, and
E, respectively, and
bold arrows identify some proteins of equal abundance in
both experiments.
E-regulated
Genes--
We sought to identify
E-regulated genes by
searching for promoter sequences that activate transcription under heat
shock conditions (43 °C). The
Mu53-lacZ
(KanR) transposon inserts randomly into the chromosome of
E. coli and generates fusions of a promoterless
lacZ reporter with regulatory sequences flanking the
insertion sites. 50,000
Mu53-lacZ (KanR)
E. coli MC4100 mutants were screened by growing colonies at 43 °C on MacConkey plates with kanamycin (Fig.
2). 1,000 mutant red colonies were picked
and pooled for further analysis. Our initial screen could not
distinguish between RpoE-regulated promoters and those that are
transcribed by other polymerases. 1,000
Mu53-lacZ (KanR) insertions were transduced into E. coli
strain SR3206 (surA::Tn10) using
bacteriophage P1. Transductants were selected for growth at 30 °C on
MacConkey agar containing kanamycin. surA encodes a
periplasmic chaperone required for folding of outer membrane proteins
(8, 30, 31). E. coli surA mutants express the
E-regulated promoters htrA and
rpoEP2 constitutively, even when cells are grown on agar
medium at 30 °C (8). 200 transposon transductants of E. coli SR3206 formed red colonies at 30 °C. These
Mu53-lacZ (KanR ) insertions were analyzed
further and transduced into E. coli strain SR3323 containing
RseA encoded on a high copy number plasmid. Overproduction of RseA
reduces E
E RNA polymerase transcription because the
anti-sigma factor sequesters
E in the cytoplasmic
membrane compartment. 78 E. coli
Mu53-lacZ transductants formed white colonies on MacConkey agar. The site of
transposon insertion in these strains was determined by DNA sequence
analysis.
Mu53-lacZ insertions identified nine
E-regulated genes: htrA, fkpA,
cutC, nlpB, purA, mdoG,
mdoH, yggN, and ytfJ. yggN and
ytfJ were identified previously by genome sequencing; however, a physiological role of these genes has not yet been described. Henceforth, we refer to these genes as ecfF
(yggN) and ecfJ (ytfJ), for
extracytoplasmic function genes F and J.
View larger version (28K):
[in a new window]
Fig. 2.
Genetic strategy using transposon mutagenesis
to search for E-regulated
genes. 50,000
Mu53-lacZ (KanR) E. coli MC4100 mutants were screened by growing colonies at 43 °C
on MacConkey plates with kanamycin. 1,000 red colonies were pooled
(filled circles on the agar plate drawing) and transduced
into E. coli MC4100
surA::Tn10. 200 colonies that developed
a red staining on MacConkey agar were transformed with a plasmid
overexpressing rseA. 78 colonies that developed white
staining on MacConkey agar were analyzed further by characterizing the
transposon insertion.
E-regulated
Genes--
Our transposon insertion mutagenesis cannot identify
E-regulated genes that are essential for E. coli growth. To identify all RpoE-regulated genes, even those that
are essential, 0.8-1.2-kilobase pair DNA fragments were generated by
Sau3A digestion of the E. coli chromosome and
cloned into pFZY digested with BamHI. pFZY is a single copy
F factor plasmid containing a promoterless lacZ gene
downstream of the BamHI site (22). Recombinant plasmids containing transcriptionally active promoter fusions were selected by
transformation of E. coli MC4100
(lacZ
). Transformants were plated on lactose
minimal agar at 30 °C. 200,000 Lac+ colonies were
replica plated on MacConkey agar and incubated at 43 °C (Fig.
3). 10,000 colonies displayed red
staining (Lac+) under heat shock conditions, representing
possible
E-regulated promoter fusions. The red colonies
were pooled and plasmids purified and transformed into E. coli SR3206 (surA::Tn10). Transformants were plated on MacConkey agar at 30 °C and screened for a red colony phenotype, consistent with
surA-dependent induction of
E-transcribed promoters. 10,000 red colonies were pooled
and made competent for transformation with pSR3323, a high copy number plasmid encoding rseA+. Transformants were
plated on MacConkey agar at 30 °C. 350 white colonies were picked.
Plasmids were extracted from the pool and used to transform strain
SR1502. E. coli SR1502 is an MC4100 variant carrying a
mutation in the rpoE gene (rpoER178G) which
displays a temperature-sensitive growth phenotype because the mutant of E
E polymerase cannot adequately transcribe
E-regulated genes (16). To determine whether promoter
fusions were transcribed by E
E polymerase, SR1502
transformants were plated on MacConkey agar at 30 °C. 500 transformants formed white colonies on MacConkey agar at 30 °C.
Plasmids were purified from these strains and analyzed by dot blot
analysis for hybridization with three radiolabeled RpoE promoter probes
(htrA, rpoEP2, and rpoHP3), revealing
fusion of the known htrA, rpoE, or
rpoH promoter to lacZ. 120 plasmids that failed
to hybridize in the dot blot experiment were analyzed by restriction
mapping and DNA sequence analysis, which identified 22 distinct
promoters. Results described below revealed that 19 of the 22 promoters
are transcribed in an E
E-dependent manner.
The promoters activate transcription of cutC, dsbC, fkpA, htrM, mdoG,
nlpB, ostA, rpoD, skp,
ecfA (f288), ecfD (yfiO), ecfE
(yaeL), ecfF (yggN), ecfG (htrG), ecfH
(yraP), ecfI (yidQ), ecfJ (ytfJ), ecfK
(UP0), and ecfL (yqjA). Genes without a previously
assigned physiological function are referred to as ecf, for
extracytoplasmic function genes.
View larger version (29K):
[in a new window]
Fig. 3.
Genetic strategy using pFZY promoter probe
vector to search for E-regulated
genes. E. coli MC4100 transformants of a promoter
library in pFZY were isolated by growing colonies at 30 °C on
lactose minimal agar. 200,000 colonies were replica plated and grown at
43 °C on MacConkey agar. 10,000 red colonies (filled
circles on the agar plate drawing) were pooled and plasmid
isolated and transformed into E. coli MC4100
rseA::Tn10. Colonies that developed red
staining after growth on MacConkey agar at 30 °C were pooled and
transformed with a plasmid overproducing RseA. 358 white colonies were
isolated and plasmids purified and transferred into E. coli
MC4100 rpoER178G. 500 colonies that developed white staining
when grown on MacConkey at 30 °C were picked and analyzed for
promoter content using Southern hybridization (htrA,
rpoE, and rpoH promoter probes) and DNA
sequencing.
E-transcribed Promoters Are Regulated by the rpoE
rseA Operon--
To quantify transcriptional regulation of
E
E polymerase-transcribed promoters, fusions were
inserted into attB (bacteriophage
attachment site) of
E. coli MC4100 and SR3206
(surA::Tn10) using
RS45 as cloning
vector (Fig. 4A).
RS45
lysogens were grown in Luria broth to A595
0.5-0.7, and LacZ activity was measured in a spectrophotometer using
o-nitrophenyl
-D-galactopyranoside as
a substrate. htrA promoter activity was monitored as a
control for a known
E-regulated gene. When
lysogens
of the surA mutant strain SR3206 were examined at 30 °C,
htrA promoter activity was increased by 4-fold compared with
wild-type E. coli. All 19 isolated promoters displayed a
similar phenotype with a 2-4-fold increase of reporter transcription
in the surA mutant strain (cutC, dsbC,
fkpA, htrM, mdoG, nlpB,
ostA, rpoD, skp, ecfA,
ecfD, ecfE, ecfF, ecfG,
ecfH, ecfI, ecfJ, ecfK, and
ecfL fusions to lacZ) (Fig. 4A). When
E. coli SR1502 (rpoER178G) was lysogenized with
RS45 derivatives, the promoter fusions expressed the LacZ reporter
only with background activity (Fig. 4B). Transformation of
SR1502 harboring a htrA-lacZ insertion with a plasmid
encoding wild-type rpoE led to a 20-fold increase in
expression of LacZ reporter. All 19 promoter fusions behaved similarly
(Fig. 4B). Increased expression was also observed in the
presence of wild-type RpoE (between 10- and 30-fold). As a final test
to determine whether the isolated promoters are transcribed by
E
E polymerase, E. coli MC4100 carrying
insertions of
RS45 derivatives were transformed with three different
plasmids: pRS3323 (overexpression of wild-type RseA), pRS3076
(overexpression of RseA
28, a mutant lacking the first 28 amino
acids), or pET-24d (control vector lacking RseA) (Fig. 4C).
Compared with strains carrying the vector alone, all transformants
expressing wild-type rseA transcribed between 10 and 60% of
lacZ reporter gene. In contrast, expression of the mutant
rseA allele (rseA
28) caused no reduction in
reporter transcription. Together these data indicate that the 20 isolated gene promoters are transcribed by E
E RNA
polymerase in a manner that is also subject to regulation by RseA. With
the exception of rpoD, the promoters were isolated multiple
times, suggesting that our search for RpoE-regulated genes has been
nearly exhaustive. Comparison of the number of isolated gene promoters
with the number of
E-regulated protein spots identified
by two-dimensional gel electrophoresis corroborates this view further.
The search for E
E-transcribed promoters identified 24 new promoters. At least 12 of the genes transcribed by
E
E polymerase encode lipoproteins or membrane proteins.
These hydrophobic proteins will not be identified by two-dimensional
gel electrophoresis.
View larger version (29K):
[in a new window]
Fig. 4.
Transcriptional activity of the selected
E-dependent promoter fused
to lacZ. Bacterial cells containing a single copy
lacZ promoter fusions (inserted with lambda vector at
attB) were assayed for promoter activity by measuring
-galactosidase expression. Panel A, promoter activities
as assayed in E. coli MC4100 wild-type (empty
bars) and surA::
Cm mutant cells
(gray bars). Panel B, promoter activities as
assayed in E. coli MC4100 carrying the chromosomal
rpoER178G mutation (partial loss-of-function mutant of
E) and the pEAD vector with (gray bars) or
without the wild-type rpoE gene (empty bars).
Panel C, promoter activities as assayed in E. coli MC4100 transformed with pET-24d (empty bars) or
pET-24d derivatives overexpressing wild-type rseA
(black bars) or rseA-
28 (gray
bars), encoding a truncated form of RseA which lacks a
E binding domain.
E-transcribed
Promoters--
Promoter sequences were subjected to a BLAST search
against the E. coli genome data bank, thereby identifying
the entire gene sequences (Table II).
Primers were designed to map transcriptional start sites. Total
mRNA was purified and used as a template for reverse transcriptase
reactions primed with specific oligonucleotides, and the generated data
are summarized in Table III. In all cases examined, the transcriptional start sites are spaced appropriately with
respect to
10 and
35 recognition sites. Comparison of DNA sequences
allowed the identification of a presumed canonical E
E
recognition site. YCTGA is positioned 7-9 nucleotides upstream of the
transcriptional start site (
10). A string of 2-6 purine nucleotides,
most often containing the sequence GAA, is positioned 16 nucleotides
upstream of the
10 site (
35 site). Some E
E promoters
transcribe operons. The first gene of these operons is htrM,
mdoG, ostA, skp, ecfA, or
ecfL, respectively (Table II). Other E
E
promoters presumably represent only one out of several other promoters
that are transcribed by other RNA polymerases (Table IV). Close examination of the skp
lpxD lpxA fabZ operon revealed the presence of an additional
E
E-dependent start site located in front of
lpxD (Tables II and III). Among the 19 promoter fusions
isolated, 4 identified E
E-dependent internal
start sites located in front of the dsbC, nlpB,
rpoD, and ecfE genes, respectively. Each of these
genes is located within a structural operon (Table II).
Function and genetic organization of genes transcribed by
EE
E-dependent promoter; in all cases putative or
known promoters lie to the left of the leftmost genes.
Sequence alignment of EE-dependent promoters
10 and
35 regions of the promoters are depicted
in bold. The +1 transcriptional start site is underlined. Some genes
contain multiple promoters, only the
E-dependent promoter is shown here (e.g.
htrMP4).
E-transcribed genes containing canonical CpxR-binding
boxes
E Regulon Are Also Regulated by
the CpxA CpxR Proteins--
CpxAR is a two-component regulatory system
that signals environmental stresses and accumulation of unfolded
polypeptides in the envelope of E. coli (16, 32). The
phosphorylated response regulator CpxR binds to specific promoter
sequences and activates transcription by E
70 RNA
polymerase. Members of the CpxAR regulon include htrA,
dsbA (periplasmic disulfide oxidant), and rotA
(periplasmic peptidyl isomerase) (4). To determine whether
transcriptional regulation by the CpxA CpxR proteins occurred in
vivo, the activity of all 20 isolated promoters was measured in
wild-type and cpxR mutant strains. cpxP promoter
activity was monitored as a control for a known CpxR-regulated gene
(4). cpxP promoter activity was decreased by 30% in the
cpxR mutant strain. The phosphatase PrpA modulates the
activity of CpxAR (28), and overproduction of PrpA led to an 80%
increase of cpxP promoter activity. Of the 20 promoters
examined here, dsbC, skp, and ecfI
behaved similarly to the cpxP promoter (Fig.
5), whereas all other promoters showed no
effect when analyzed in cpxR mutant strains (data not
shown). The CpxR binding site has been identified as tandem repeats of the nucleotide sequence GGTNANY. The dsbC, skp,
and ecfI promoter sequences were found to harbor DNA repeat
elements that matched the consensus sequence of CpxR binding sites
(Table IV).
View larger version (16K):
[in a new window]
Fig. 5.
Regulation by the CpxR transcriptional
response regulator. Bacterial cells containing a single copy
lacZ promoter fusions (inserted with lambda vector at
attB) were assayed for promoter activity by measuring
-galactosidase expression. Promoter activities were assayed in
E. coli MC4100 (wild-type, empty bars), a
cpxR mutant derivative (black bars), or a
prpA-overexpressing strain (gray bars).
-Galactosidase activities were unaffected when E. coli
MC4100 was transformed with the empty vector control alone (data not
shown).
E-regulated Genes That Are Essential for E. coliGrowth--
E
E represents a minor RNA polymerase
species and transcribes only 20 of the 4,500 genes encoded by the
genome of E. coli. We wondered why rpoE may be
essential for E. coli growth. Two E
E-transcribed genes encode sigma factors for major RNA
polymerase species, rpoD
(
2
'
70) and rpoH
(
2
'
32). rpoD is
essential for growth at all temperature. It seems unlikely that
E
E transcription of rpoD is essential for
E. coli growth because rpoD is transcribed by
multiple promoters and RNA polymerase species. The rpoH gene
is also transcribed by several RNA polymerase species, and
E
E recognizes only one of the three known promoters.
Deletion of rpoH is tolerated at elevated temperature upon
overexpression of groEL and dnaK operon genes
(33). Thus, if E
E transcription of rpoD and
rpoH is not essential, can RpoE-mediated transcription of
some other genes be required for E. coli growth?
elements and cloned on a plasmid carrying a
temperature-sensitive replicon and the sacB marker. After
transformation of plasmids into E. coli MC4100, single
crossover recombination events with wild-type ecf sequences
were isolated by plating bacteria at 43 °C, a condition that stalls
plasmid replication, and by selecting for plasmid-encoded
chloramphenicol transferase activity on Luria agar supplemented with
chloramphenicol. The resulting plasmid cointegrates into the E. coli chromosome are merodiploid and contain two copies of the
ecf gene under study, a wild-type and a mutant allele.
Growth of cointegrate strains on sucrose-containing media serves as a
counterselection for plasmid-encoded sacB because as
expression of the sacB gene product leads to the
accumulation of toxic metabolites during sucrose fermentation. Thus,
when cointegrated strains are streaked on agar medium containing
sucrose as well as antibiotic selection for the
element, the
resulting colonies represent ecf mutants arising from double
crossover recombination. Using this experimental scheme,
ecfA, ecfD, ecfF, ecfH,
ecfI, ecfJ, and ecfK knockout mutants
were obtained. A Tn10 insertional knockout mutation of
ecfG had been isolated
previously.2 Two of the
isolated knockout mutant strains, ecfG and ecfJ,
displayed a temperature-sensitive growth phenotype above 43 °C.
E-regulated Genes--
Folding of polypeptides in the
bacterial cytoplasm is catalyzed by many factors that fulfill partially
redundant functions. GroEL-GroES are essential for E. coli
growth. DnaK, a member of the Hsp70 family, is nonessential for
E. coli growth; however, cells cannot tolerate the
simultaneous loss of DnaK and trigger factor (a cytoplasmic peptidyl
isomerase encoded by the tig gene) because these catalysts
are required for the folding of newly synthesized polypeptides (34,
35). When tested in the experimental scheme for knockout mutations
described above, tig mutants can be obtained in a wild-type
E. coli strain but not in a mutant lacking the
dnaK gene; this phenotype is referred to as synthetic lethal. In other cases, deletion of a single gene may produce no
phenotype; however, deletion of two genes whose products act on the
same pathway may restrict viability and growth of E. coli cells, causing a synthetic conditional lethal phenotype at elevated temperatures. We tested various mutant strains carrying deletions of
E-regulated genes for a synthetic phenotype. Some of the
relevant data are reported in Table V.
E. coli cells cannot tolerate the loss of genes encoding two
main periplasmic folding factors, skp and fkpA.
Double mutants dsbC/htrA,
skp/htrA, and fkpA/htrA
display a synthetic conditional lethal phenotype. Thus, at elevated
temperatures E. coli cells require HtrA protease to remove
misfolded polypeptides in the periplasm, a condition that is aggravated
when specific folding catalysts are nonfunctional. Loss of both
fkpA and surA also leads to a synthetic
conditional lethal phenotype. When combined with htrA, the
triple mutant fkpA/surA/htrA is nonviable (Table V).
Phenotypic analysis of members of the E regulon
E Regulon--
The
E regulon can be viewed as providing essential folding
functions for proteins that are located in the bacterial envelope. An
increase in temperature (38-45 °C) weakens the interactions that
maintain the three-dimensional structure of polypeptides at
physiological temperature (25-37 °C): hydrogen bonds, ion bonds as
well as van der Waal's forces. Other conditions that alter the above
mentioned parameters of protein folding and stability should therefore
also induce the
E regulon. We wondered whether a
reduction in temperature (14-24 °C) could induce the
E regulon. E. coli cold shock appears to be a
regulated response requiring many genes; however, a specific sensing or
transcriptional regulatory mechanism has thus far not been established.
Using MC4100 strains carrying single copy insertions of
E-regulated promoters fused to lacZ, we
observed about a 20-30% increase in transcription after incubating
cells for 1 h at 14 °C (Fig. 6).
Thus, rapid reduction of ambient temperature also stimulates
E
E polymerase, causing a small increase in the
expression of folding catalysts.
View larger version (22K):
[in a new window]
Fig. 6.
The E
regulon is cold shock-inducible. Bacterial cells containing a
single copy lacZ promoter fusions (inserted with lambda
vector at attB) were assayed for promoter activity by
measuring
-galactosidase expression. Promoter activities were
assayed in E. coli MC4100 at 37 °C (empty
bars) or 14 °C (gray bars).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E RNA polymerase is thought to be dedicated to
expressing folding catalysts that act on proteins in the bacterial
envelope. Here we measured the size of the
E regulon
with two methods: two-dimensional gel electrophoresis of RpoE-induced
cells and cloning of RpoE-regulated promoters. Results from both
experiments as well as previous work suggest that E
E
transcribes some 43 genes. We describe here 20 new promoters that are
recognized by E
E RNA polymerase. Some of the genes
regulated by E
E were hitherto unknown and have been
designated ecf, for extracytoplasmic encoding function. Some
of the encoded gene products are located in the periplasmic space and
act directly on misfolded proteins: DsbC, FkpA, HtrA, Skp, and SurA.
Some other gene products are located in the bacterial cytoplasm and
serve regulatory functions that coordinate the expression of the
E regulon with environmental conditions. RpoE, RpoH, and
RpoD represent components of various RNA polymerase species, whereas
RseA, RseB, and RseC regulate the availability of
E for
core RNA polymerase. Several
E-regulated gene products
are involved in the synthesis of lipopolysaccharide, a component of the
outer membrane of Gram-negative bacteria. Lipopolysaccharide has been
proposed to act as a cofactor for the membrane assembly of outer
membrane proteins, a pathway that appears to require Skp activity (8).
Skp has also been shown to play other roles in envelope assembly (36).
It seems noteworthy however that skp mutant cells contain
increased amounts of lipopolysaccharide within the periplasm (36). It
is as if deletion of the presumed folding factor (Skp) may lead to the
simultaneous accumulation of its cofactor (lipopolysaccharide). The
rfaDFCL and lpxDA genes provide known components
of the lipopolysaccharide biosynthetic pathway and are transcribed by
E
E polymerase. In fact, the lpxD lpxA fabZ
genes are regulated by two
E-dependent
promoters: one placed in front of skp (the first gene of the
operon) and a second one in front of lpxD. Our preliminary results suggest that the ecfABC gene products may also be
involved in the lipopolysaccharide biosynthetic
pathway.3
E-regulated genes encode proteins with sensory
functions. MdoG is involved in coordinating cellular pressure with the biosynthesis of periplasmic membrane-derived oligosaccharides (37),
whereas CutC has been postulated to be involved in copper homeostasis
(38). The requirement of these gene products for protein folding in the
periplasmic space is not immediately apparent. In this and perhaps in
other cases, the presence of a
E promoter may provide
growth advantages for the E. coli host which are not related
to protein folding. The largest group of
E-regulated
genes encodes proteins located in the inner (NlpB, EcfD, EcfG, EcfI,
and EcfL) and outer membranes (EcfK and EcfM). The precise function of
these proteins remains to be established; however, it is conceivable
that the membrane proteins act directly on misfolded membrane proteins
and promote either polypeptide degradation or insertion into the lipid
bilayer. Alternatively, membrane proteins may be involved in the
transport and assembly of lipopolysaccharide into the physiological
bilayer structures.
E regulon is essential for
E. coli growth. EcfE appears to be a member of a large group
of proteases designated RIP (regulated intramembrane proteolysis).
Proteases of the RIP family are needed for diverse functions such as
lipid metabolism, cell differentiation, and response to unfolded
proteins (39, 40). We are currently investigating the role of EcfE in
signaling envelope stress in E. coli.
E regulon has evolved to control at
least two cellular processes, folding of polypeptides in the bacterial envelope and biosynthesis/transport of lipopolysaccharide. Conditions that cause unfolding of polypeptides are signaled by the RseA and RseB
proteins (11). It is conceivable that the
E regulon can
sense and respond to changes in lipopolysaccharide metabolism. Our
future work will address this possibility.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank O. Schneewind (UCLA) for a critical review of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Fond National Scientifique Suisse Grant FN3100-059131.99/1 (to S. R.) and United States Public Health Service Grant GM58266 (to D. M.).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.
¶ To whom correspondence should be addressed: Dept. of Microbiology, Immunology, and Molecular Genetics, UCLA, 609 Charles Young Dr., Los Angeles, CA 90095. E-mail: missiaka@microbio.ucla.edu.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M100464200
2 S. Raina, unpublished data.
3 C. Dartigalongue, D. Missiakas, and S. Raina, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
E and
2
', core RNA polymerase;
RpoE and
E, sigma E transcription factor;
E
E and
2
'
E, holoenzyme complexed to sigma
E;
Rse, regulator of
E;
28RseA, a variant of RseA
lacking the first 28 amino acids;
rpoER178G, an allele of
rpoE encoding a mutant of
E with severely
impaired transcriptional activity;
Ecf, extracytoplasmic function gene
product.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gross, C. A. (1996) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F. C. , Curtis, R. I. , Ingraham, J. L. , Lin, E. C. C. , Low, K. B. , Magasanik, B. , Reznikoff, W. S. , Riley, M. , Schaechter, M. , and Umbarger, H. E., eds) , pp. 1382-1399, American Society for Microbiology, Washington, D. C. |
2. | Missiakas, D., Raina, S., and Georgopoulos, C. (1996) in Regulation of Gene Expression in Escherichia coli (Lin, E. C. C. , and Lynch, S. A., eds) , pp. 481-501, R. G. Landes Company, Austin, TX |
3. | Missiakas, D., and Raina, S. (1998) Mol. Microbiol. 28, 1059-1066[CrossRef][Medline] [Order article via Infotrieve] |
4. | Raivio, T. L., and Silhavy, T. J. (1999) Curr. Opin. Microbiol. 2, 159-165[CrossRef][Medline] [Order article via Infotrieve] |
5. | Lonetto, M., Brown, K. L., Rudd, K. E., and Buttner, M. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7573-7577[Abstract] |
6. |
Kang, J. G.,
Paget, M. S.,
Seok, Y. J.,
Hahn, M. Y.,
Bae, J. B.,
Hahn, J. S.,
Kleanthous, C.,
Buttner, M. J.,
and Roe, J. H.
(1999)
EMBO J.
18,
4292-4298 |
7. | Mecsas, J., Rouvière, P. E., Erickson, J. W., Donohue, T. J., and Gross, C. A. (1993) Genes Dev. 7, 2618-2628[Abstract] |
8. | Missiakas, D., Betton, J.-M., and Raina, S. (1996) Mol. Microbiol. 21, 871-884[Medline] [Order article via Infotrieve] |
9. | de las Peñas, A., Conolly, L., and Gross, C. A. (1997) Mol. Microbiol. 24, 3373-3386 |
10. | Missiakas, D., Mayer, M., Lemaire, M., Georgopoulos, C., and Raina, S. (1997) Mol. Microbiol. 24, 355-371[Medline] [Order article via Infotrieve] |
11. |
Collinet, B.,
Yuzawa, H.,
Chen, T.,
Herrera, C.,
and Missiakas, D.
(2000)
J. Biol. Chem.
275,
33898-33904 |
12. |
Ades, S. E.,
Connolly, L. E.,
Alba, B. M.,
and Gross, C. A.
(1999)
Genes Dev.
13,
2449-2461 |
13. | Strauch, K. L., and Beckwith, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1576-1580[Abstract] |
14. | Erickson, J. W., and Gross, C. A. (1989) Genes Dev. 3, 1462-1471[Abstract] |
15. | Danese, P. N., and Silhavy, T. J. (1997) Genes Dev. 11, 1183-1193[Abstract] |
16. | Raina, S., Missiakas, D., and Georgopoulos, C. (1995) EMBO J. 14, 1043-1055[Abstract] |
17. | Rouvière, P., de las Peñas, A., Mecsas, J., Lu, C. Z., Rudd, K. E., and Gross, C. A. (1995) EMBO J. 14, 1032-1042[Abstract] |
18. | de las Peñas, A., Connolly, L., and Gross, C. A. (1997) J. Bacteriol. 179, 6862-6864[Abstract] |
19. | Miller, J. (1992) A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
20. | Missiakas, D., Georgopoulos, C., and Raina, S. (1993) J. Bacteriol. 175, 2613-2624[Abstract] |
21. | Bremer, E., Silhavy, T. J., and Weinstock, G. M. (1985) J. Bacteriol. 162, 1092-1099[Medline] [Order article via Infotrieve] |
22. | Koop, A. H., Hartley, M. E., and Bourgeois, S. (1987) Gene (Amst.) 52, 245-256[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Dartigalongue, C.,
and Raina, S.
(1998)
EMBO J.
17,
3968-3980 |
24. | Simons, R. W., Houman, F., and Kleckner, N. (1987) Gene (Amst.) 53, 85-96[CrossRef][Medline] [Order article via Infotrieve] |
25. | Wang, R. F., and Kushner, S. R. (1991) Gene (Amst.) 100, 195-199[CrossRef][Medline] [Order article via Infotrieve] |
26. | Fellay, R., Frey, J., and Krisch, H. (1987) Gene (Amst.) 52, 147-154[CrossRef][Medline] [Order article via Infotrieve] |
27. | Link, A. J., Phillips, D., and Church, G. M. (1997) J. Bacteriol. 179, 6228-6237[Abstract] |
28. |
Missiakas, D.,
and Raina, S.
(1997)
EMBO J.
16,
1670-1685 |
29. | Neidhardt, F. C., and VanBogelen, R. A. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F. C. , Curtis, R. I. , Ingraham, J. L. , Lin, E. C. , Low, K. B. , Magasanik, B. , Reznikoff, W. S. , Riley, M. , Schaechter, M. , and Umbarger, H. E., eds) , pp. 1334-1345, American Society for Microbiology, Washington, D. C. |
30. | Lazar, S. W., and Kolter, R. (1996) J. Bacteriol. 178, 1770-1773[Abstract] |
31. | Rouvière, P., and Gross, C. A. (1996) Genes Dev. 10, 3170-3182[Abstract] |
32. | Danese, P., Snyder, W. B., Cosma, C., Davis, L. J., and Silhavy, T. J. (1995) Genes Dev. 9, 387-398[Abstract] |
33. | Kusukawa, N., and Yura, T. (1988) Genes Dev. 2, 874-882[Abstract] |
34. | Teter, S. A., Houry, W. A., Ang, D., Tradler, T., Rockabrand, D., Fischer, G., Blum, P., Georgopoulos, C., and Hartl, F. U. (1999) Cell 97, 755-765[Medline] [Order article via Infotrieve] |
35. | Deuerling, E., Schulze-Specking, A., Tomoyasu, T., Mogk, A., and Bukau, B. (1999) Nature 400, 693-696[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Schafer, U.,
Beck, K.,
and Muller, M.
(1999)
J. Biol. Chem.
274,
24567-24574 |
37. | Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1092-1095[Abstract] |
38. |
Blattner, F. R.,
Plunkett, G.,
Bloch, C. A.,
Perna, N. T.,
Burland, V.,
Riley, M.,
Collado-Vides, J.,
Glasner, J. D.,
Rode, C. K.,
Mayhew, G. F.,
Gregor, J.,
Davis, N. W.,
Kirkpatrick, H. A.,
Goeden, M. A.,
Rose, D. J.,
Mau, B.,
and Shao, Y.
(1997)
Science
277,
1453-1474 |
39. | Niwa, M., Sidrauski, C., Kaufman, R. J., and Walter, P. (1999) Cell 99, 691-702[Medline] [Order article via Infotrieve] |
40. | Brown, M. S., Ye, J., Rawson, R. B., and Goldstein, J. L. (2000) Cell 100, 391-398[Medline] [Order article via Infotrieve] |
41. | Missiakas, D., Georgopoulos, C., and Raina, S. (1994) EMBO J. 13, 2013-2020[Abstract] |
42. | Raina, S., and Georgopoulos, C. (1991) Nucleic Acids Res. 19, 3811-3819[Abstract] |
43. | Lipinska, B., Fayet, O., Baird, L., and Georgopoulos, C. (1989) J. Bacteriol. 171, 1574-1584[Medline] [Order article via Infotrieve] |