Unité de Microbiologie et
Génétique, UMR CNRS 5122, Université Lyon 1, F-69622 Villeurbanne Cedex, France
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INTRODUCTION |
In Escherichia coli, the
level of expression of many genes is influenced by the pH of the
growth medium (pHo) (11, 19). E. coli has a
constitutive homeostatic mechanism which allows cells to maintain their
internal pH between 7.4 and 7.8 over a pHo range of 5 to 8.5 (35). Within this pHo range, cells are able to sense a
stimulus, triggered by the pHo, and transmit it to the target genes.
Among these target genes are the maltose and the porin regulons
(1, 12, 14).
ompF and ompC porin genes are controlled by the
cognate sensor kinase EnvZ and the response regulator OmpR
(22). A higher OmpR phosphate level at low pHo than at
high pHo could be responsible for the pHo regulation of porin
genes (14). In addition, the alternate phosphodonor
acetyl phosphate may play a crucial role in the modulation of the
OmpR-phosphate level and the subsequent pHo regulation of porin genes
(14).
The maltose regulon of E. coli consists of genes encoding
proteins involved in the uptake and metabolism of maltose and
maltodextrins (4, 5). These genes are clustered in five
transcriptional units controlled by the transcriptional activator MalT.
malT expression is subjected to catabolite repression and
then requires the presence of the cyclic AMP (cAMP)-cAMP receptor
protein (CRP) complex. Recently, Mlc, a global negative regulator for
the transcription of several genes whose products are involved in
carbon utilization, has been found to be a repressor for
malT (6). The expression of some operons and
genes of the maltose regulon is also directly under the control of the
cAMP-CRP complex. Lowering the pHo from 8 to 5 decreases
malTp activity. This pHo effect relies on the cAMP-CRP
binding (1). Indeed, with a cAMP-CRP-independent
malT promoter, the pHo effect is not observed.
malT pHo regulation triggers the pHo regulation of all the
MalT-dependent promoters. In the absence of Mlc, the pHo regulation of
malT is still effective (1). Recently, Eppler
and Boos demonstrated that growth in tryptone broth containing glycerol
reduced malT expression two- to threefold compared to
tryptone broth without glycerol (10). To establish this
repression, the enzyme IIAGlc, the cAMP-CRP
complex, and the phosphorylation of glycerol to sn-glycerol-3-phosphate (G3P) by glycerol kinase (GlpK) are
all necessary, but further metabolism to dihydroxyacetone by glycerol phosphate dehydrogenase is not (10).
The glycerol regulon is organized in multiple loci around the
chromosome, and its expression is negatively controlled by GlpR (17). The glpTQ and glpACB operons
located near 51 min encode the G3P
permease/glycerophosphodiesterase and the subunits of the
anaerobic G3P dehydrogenase, respectively (9). The
glpD gene, encoding the aerobic G3P dehydrogenase, is
located near 77 min and is transcribed divergently from the
glpEGR operon (27). GlpE is a sulfur
transferase (23). glpG encodes a protein of unknown function, and glpR encodes the repressor (28,
34). The glpFKX operon located near 88 min encodes
the glycerol diffusion facilitator (GlpF), the glycerol kinase (GlpK),
and a fructose 1,6-biphosphatase (GlpX) (7). This operon
is subjected to multiple controls, including catabolite repression
mediated by cAMP-CRP and repression by cooperative binding of GlpR to
tandem operator sites which overlap the promoter. G3P, the product of
the reaction catalyzed by glycerol kinase, is the inducer of the
glp regulon (17). GlpK, as well as MalK, is
involved in interactions with the unphosphorylated form of enzyme
IIAGlc, which is an intermediate in the
phosphorylation cascade of the phosphotransferase (PTS)-mediated
uptake and concomitant phosphorylation of glucose. This
interaction inhibits the metabolism of non-PTS-carbohydrates, such
as glycerol and maltose, by preventing the induction of their respective catabolic operons (21). This process is
known as inducer exclusion (15). Rohwer et al. have shown
that a high level of glycerol kinase could result in
IIAGlc sequestration into an inactive complex
(25). The phosphorylated form of
IIAGlc is considered to be involved in the
activation of adenylate cyclase, and this leads to increased
intracellular levels of cAMP, which binds to CRP and elaborates the
cAMP-CRP complex (24).
As the involvement of the cAMP-CRP complex in the
glycerol-dependent repression of malT transcription in rich
medium (10) and in the pHo regulation of malT
transcription during growth with glycerol as a carbon source
(1) has been clearly established, we were interested in
studying the connections between the glycerol effect and the pHo
regulation of maltose regulon. This study shows that the
phosphoenolpyruvate:carbohydrate PTS system and adenylate cyclase are
involved in the complex molecular interplay between the different
regulatory mechanisms that regulate gene expression according to
the pHo.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
Bacterial strains
and plasmids used in this study are listed in Table
1. Standard media were used in this
study, including Luria-Bertani (LB) broth (18), MacConkey
medium with lactose (final concentration, 2% [wt/vol]) added (Difco
Laboratories, Detroit, Mich.), and M63 minimal medium (18)
supplemented with 3 µM thiamine hydrochloride, the appropriate amino
acids, and sugars as the carbon source. Buffered minimal medium (MM)
was as previously described (13). It was adjusted with 1 N
NaOH to pH 4.75, 5, 5.25, 5.5, 5.75, 6.5, 7, 7.5, and 8. MM-MES is a
solid MM containing 100 mM MES [2-(N-morpholino)ethane
sulfonic acid; pKa, 6.1], instead of 50 mM in MM
and no TAPS [tris(hydroxymethyl)methylaminopropane sulfonic acid;
pKa, 8.4]. This buffered medium was adjusted to pH 5 with 1 N NaOH. MM and MM-MES were supplemented with 3 µM thiamine hydrochloride. Carbon sources used in these media were 44 mM
glycerol, 12 mM G3P, 21 mM succinate, 22 mM glucose, and 58 mM maltose.
When required, 40 µg of X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactoside) ml
1 or the following antibiotics were added: 30 µg (pJEL derivatives) or 100 µg of ampicillin
ml1, 50 µg of kanamycin
ml1, 15 µg of tetracycline
ml1, and 20 µg of chloramphenicol
ml1.
Growth conditions.
Overnight subcultures, grown at 30°C in
LB broth supplemented with the appropriate antibiotics, were used to
inoculate MM adjusted to different pHo values and supplemented with
various carbon sources and antibiotics. These cultures were incubated at 30°C until the stationary phase was reached. At this point, the
different buffered cultures were diluted with the same fresh medium to
an optical density at 600 nm of 0.02 (path length, 1 cm; Jouan
spectrophotometer). These new cultures were incubated at 30°C up to
an optical density at 600 nm between 0.2 and 0.3. Up to this cell
density, the pHo remains constant.
Enzyme assays.
-Galactosidase activities associated with
operon or gene fusions were measured on toluenized cells, as previously
described (12). One unit of -galactosidase activity was
defined as the amount of enzyme that hydrolyzed 1 nmol of substrate
min1. Results from at least three independent
experiments were averaged to obtain the values presented below.
PCR amplification.
PCRs were performed using standard
conditions (16) and Pfu enzyme (Promega Corp,
Madison, Wis.). Primers were phosphorylated by the T4 polynucleotide
kinase prior to amplification. The amplified fragments were separated
by agarose gel electrophoresis, extracted (QiaEx gel purification kit;
Qiagen), and ligated into the appropriate vector. The ligated DNA was
transformed into E. coli DH5
, and antibiotic-resistant
clones were isolated by growth at 37°C in LB broth containing the
appropriate antibiotics.
In vitro plasmid construction.
Fragments of 4,374 bp,
including the glp promoter,
were amplified from MC4100 and GPH8840
chromosomes by PCR (Fig. 1A). Primers used to amplify this
fragment hybridized to a region 615 bases upstream of the
transcriptional start point (+1) of glpFKX (PrGlpF, 5'-AGATGAAGCGTAATCAGACC-3') and to a region 152 bases
downstream of the glpX last codon (A10,
5'-TGAACGGTGAAGACTAAACAG-3'). The 4,374-bp fragments
were ligated into HincII-cut and dephosphorylated pACYC177.
Kanamycin-resistant clones were isolated. pGPH9925 corresponds to
the PCR product from MC4100, and pGPH9928 corresponds to that from
GPH8840.
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FIG. 1.
Schematic representation of the glpFKX
operon cloned in pACYC177 and sequence of the parental and mutant
glp promoter regions. (A) Open reading frames within the
glpFKX operon, with the direction of transcription, are
indicated by open arrows. Restriction sites: EI, EcoRI;
BI, BamHI; R, RsrII; EV,
EcoRV; BII, BstEII; A,
AatII. The glp transcriptional start site
is indicated with an arrow upstream of glpF
(32). The primers used in PCR amplification are indicated
with triangles. (B) Grey boxes indicate the binding site of the GlpR
repressor. Striped boxes indicate the cAMP-CRP binding site. The
transcriptional start point is at position +1 (32).
Putative  10 and 35 sequences are underlined.
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The glp promoter region was amplified from MC4100 and
GPH8840 chromosomes by PCR. The primers used were PrGlpF and EGlpF
(5'-GTAGTCATATTACAGCGAAGCTT-3') (Fig. 1A), which hybridized
to a region encompassing the last codon of glpF. These
primers gave 1,577-bp products which were digested by BamHI.
The 718-bp promoter fragments were separated by agarose gel
electrophoresis, extracted, and ligated into SmaI- and
BamHI-digested pUC18. The 723-bp promoter fragments were
excised from pUC18 with EcoRI and BamHI and
ligated into EcoRI- and BamHI-digested pJEL250 to
create pGPH11247, with the parental glpFp operon fusion, and
pGPH11248, with the mutant glpFp18 operon fusion.
A 3,751-bp fragment, including the glp operon without its
promoter, was amplified from the MC4100 chromosome by PCR. Primer A10
was used in combination with primer GLP8
(5'-CATCGTGGAGCTCCGTGACTTTC-3') (Fig. 1A). This primer
hybridized to a region extending from +22 to +44 relative to the
transcriptional start point. At position +32, a C was introduced
instead of a G in order to create a SacI site for
subcloning. The 3,751-bp fragment was SacI digested, and a
fragment containing a promoterless glp operon, but with an
intact Shine-Dalgarno site, was obtained. The 3,741-bp fragment was purified and ligated into SmaI- and
SacI-digested pACT3 to create pGPH11542.
In order to test the effect of each glpFKX gene,
plasmid constructs from pGPH9925 were generated (Fig. 1A). pGPH9925 was
digested with EcoRI and EcoRV, end filled with a
Klenow fragment, and self-ligated to create pGPH11249, which
expresses only glpX. pGPH9925 was digested with
AatII and self-ligated to create pGPH11250, which
keeps only glpF and glpK intact. pGPH11250 was
digested with EcoRI and RsrII, end filled with a
Klenow fragment, and self-ligated to create pGPH11264, which contains
only glpK. pGPH11250 was digested with BstEII and self-ligated to create pGPH9993, which contains
glpF and part of glpK.
Nucleotide sequencing.
Sequencing was carried out by Genome
Express (Grenoble, France) using fluorescent dye terminator technology
and then analyzed on a Applied Biosystems 373 automated sequencer.
In vivo genetic methods.
Exponentially growing MC4100 cells
were harvested by centrifugation, washed twice, and resuspended in 0.1 M sodium citrate (pH 5.5).
N-Methyl-N'-nitrosoguanidine was added to a final
concentration of 40 µg ml1. After 12 min at
37°C without shaking, cells were centrifuged and resuspended in 100 mM phosphate buffer (pH 7.0). This suspension was used to inoculate
five independent expression cultures that were incubated at 37°C for
at least 20 h. From each culture, electrocompetent cells were
prepared and transformed with the pGPH1768 plasmid [(malK'-lacZ+)1
operon fusion]. After 1 h at 30°C, cells were centrifuged and resuspended in M63 medium. Appropriate dilutions were spread on MM-MES
(pH 5.0) plates containing ampicillin, glycerol, and X-Gal. After 3 days at 30°C, blue colonies were picked and restreaked twice on the
same medium.
Generalized transductions with P1vir were performed as
described by Miller (18). Hfr mapping was performed using
a set of Hfr Tn10 strains (31) carrying the F
plasmid at various sites scattered around the chromosome.
Curing strain from pGPH1768.
An overnight GPH8840
subculture, grown in LB broth at 30°C, was used to inoculate fresh LB
broth (103-fold dilution). These cultures were
incubated at 30°C until the stationary phase was reached. Appropriate
dilutions were spread on MacConkey medium with lactose added. Cultures
were incubated at 30°C, and white colonies among the pink ones were
picked and restreaked. The analysis showed that the white colonies did
not harbor any plasmid. Strain GPH9244 was conserved for further study.
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RESULTS |
malT and malK pHo regulation without
glpFKX induction.
The pHo regulation of the maltose
regulon with glycerol as the carbon source was described (1).
Because of the link between the mal and glp
regulons, malT transcription and that of malK, according to the pHo, were compared with and without glpFKX
expression. The glpFKX operon is not induced when succinate,
pyruvate, lactate, ribose, or maltose is used as the carbon
source, and glycerol and G3P are the carbon sources known to
induce glpFKX expression.
When glpFKX was induced in the presence of glycerol (Fig.
2), malT and malK
expressions were repressed at low pHo and reached higher levels as the
pHo was increased. With G3P as the carbon source, malT
expression was high regardless of the pHo (Fig. 2A), and
malK expression was quite high at low pHo and decreased to the level observed with glycerol as the carbon source as the pHo became
higher than 6.5 (Fig. 2B).
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FIG. 2.
pHo regulation of malT and
malK during growth with different carbon sources.
-Galactosidase activity was assayed during growth at 30°C in MM
adjusted to different pH values and supplemented with ampicillin and
the indicated carbon source. (A)
(malT'-lacZ+)1
operon fusion on pJEL250 (GPH8881); (B)
(malK'-lacZ+)1
operon fusion on pJEL250 (GPH1768). (Inset) Induced malK
expression with maltose as the carbon source. Error bars show standard
deviations. The absence of error bars indicates that the deviation fell
below the resolution limit of the graphing program.
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Without glpFKX induction, in the presence of succinate (Fig.
2), pyruvate, lactate, or ribose (data not shown), malT and
malK expression was not repressed during growth in acidic
medium. With maltose as the carbon source, malT expression
was low regardless of the pHo. Thus, with these carbon sources,
the maltose regulon was not pHo regulated. We hypothesize that
the synthesis of GlpF, GlpK, or GlpX may induce the repression of the
maltose regulon during growth in acidic medium.
The different results obtained with G3P and glycerol demonstrated that
the repression of the maltose regulon by a Glp protein may be linked to
the entry of glycerol via GlpF or to glycerol phosphorylation by GlpK.
Further metabolism of glycerol, beyond G3P, would not be involved in
maltose regulon repression during growth at low pHo.
Isolation and characterization of a mutant altered in the
glpFKX promoter.
In a mutant search aiming to
identify genes whose products are involved in the putative pHo
transduction pathway(s), we looked for mutants altered in the pHo
regulation of the malK gene. MC4100 cells were treated with
N-methyl-N'-nitrosoguanidine and then transformed with plasmid pGPH1768 carrying the
(malK'-lacZ+)1 operon
fusion. On buffered MM, adjusted to pH 5 and supplemented with X-Gal
and glycerol as the carbon source, the basal
(malK'-lacZ+)1
expression was too low for staining in blue bacterial colonies. Uninduced expression of the maltose regulon is used for expression in
MM with glycerol as the carbon source. Mutants which displayed high
expression of the fusion during growth at pHo 5 and in the absence of
maltose were visualized as blue colonies. Seventeen independent mutants
with an increased expression of malK in acidic medium
(iea) were isolated. The iea18 mutation is
described in this paper.
In the parental background of strain MC4100,
(malK'-lacZ+)1
expression (Fig. 3A) was very low at a
pHo of 4.75 and increased as the pHo reached 5.5. In the
iea18 strain, uninduced
(malK'-lacZ+)1
expression was high at low pHo and decreased to the level observed in
the parental strain at high pHo (Fig. 3A). To study the effect of
iea18 on malP and malT transcription,
strain GPH8840 was cured from pGPH1768 and transformed with pGPH9463
(strain GPH9473) or pGPH8881 (strain GPH9401), harboring, respectively,
the (malP'-lacZ+)1 or
the (malT'-lacZ+)1
operon fusion. Data from Fig. 3B show that malP promoter
activity was high whatever the pHo in the iea18 background
and in the presence of glycerol plus maltose in the growth medium. The
induced level of malP promoter activity was measured because
its uninduced expression is too low to be detected. In the
iea18 background with glycerol as the carbon source,
malT expression was as high, regardless of the pHo
(Fig. 3C), as those observed in the presence of G3P as the carbon
source (compare Fig. 3C and 2A).
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FIG. 3.
Influence of the iea18 mutation on
malK, malP, and malT pHo
regulation. -Galactosidase activity was assayed during growth at
30°C in MM adjusted to different pHs and supplemented with ampicillin
and glycerol (A and C) or glycerol and maltose (B).  , parental
strain;  , iea18 strain. (A)
(malK'-lacZ+)1
operon fusion on pJEL250 (GPH1768 and GPH8840); (B)
(malP'-lacZ+)1
operon fusion on pJEL250 (GPH9463 and GPH9473); (C)
(malT'-lacZ+)1
operon fusion on pJEL250 (GPH8881 and GPH9401). Error bars are as
in Fig. 2.
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The effect of the iea18 mutation on the transcription of
ompF and ompC porin genes was measured (Fig.
4). In the parental background, and with
glycerol as the carbon source, ompF expression was higher in
neutral than in acidic medium (Fig. 4A) whereas ompC
expression was higher at low pHo than at pHo 7 (Fig. 4B). In the
presence of the iea18 mutation, ompF
transcription was not further decreased in acidic medium, but
there was no evidence of an effect on ompC expression.
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FIG. 4.
ompF and ompC expression
at pHo 5 and 7 in parental and iea18 strains. Strains
were grown at 30°C in MM adjusted to pH 5 or 7 and supplemented with
glycerol as the carbon source. (A) GPH9604 parental strain and GPH9605
iea18 strain; (B) GPH9910 parental strain and GPH9909
iea18 strain. Error bars are as in Fig. 2.
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The iea18 mutation was associated with slower growth with
glycerol as the carbon source. Hfr and P1 transduction mapping
(31) showed that the mutation iea18 was linked
to a Tn10 marker inserted close to metF (min 89).
To determine if the iea18 mutation altered the
glpFKX operon (min 88.7), chromosomal DNA from
strains MC4100 and GPH8840 was isolated and used as a template in PCR
amplifications with the primer pair PrGlpF-A10 (Fig. 1A) to amplify the
glpFKX operon and its 5' upstream region. Wild-type and
mutant fragments of 4,374 bp were sequenced, and comparison of the
sequences enabled us to identify a single mutation located 34 bp
upstream of the transcription start site (Fig. 1B). The
iea18 mutation was renamed glpFp18. Such a
mutation, located in the 35 region of the promoter and in the GlpR-3
binding site of GlpR and close to the CRP2 binding site of cAMP-CRP
(Fig. 1B), could potentially yield an altered level of
glpFKX operon transcription.
To test this hypothesis, transcriptional fusions of lacZ as
the reporter gene and the wild-type glpFp or mutant
glpFp18 promoter were constructed. Fragments of 718 bp, from
PrGlpF to the BamHI site in glpF (Fig. 1A),
were cloned in pJEL250 to yield a wild-type (glpF'-lacZ+)1 and a
mutant
(glpFp18'-lacZ+)1
operon fusion (pGPH11247 and pGPH11248, respectively). During growth in
MM with glycerol as the carbon source, the glpFp18 mutation reduced, regardless of the pHo, the -galactosidase activity of the
corresponding fusion by a factor of 10 to 12, compared to the wild-type
promoter (Fig. 5). glpFp18 is
a promoter-down mutation of the glpFKX operon. Our results
also indicate that growth at neutral or higher pHo, with glycerol as
the carbon source, stimulated the transcription initiated at both
glpFp (Fig. 5A) and glpFp18 (Fig. 5B). Thus, the
glpFKX operon belongs to the pH stimulon.
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FIG. 5.
Parental and mutant glp promoter
activities according to the pHo. -Galactosidase activity was
assayed during growth at 30°C in MM adjusted to different pHs
and supplemented with glycerol and ampicillin. (A)
(glpF'-lacZ+)1
operon fusion on pJEL250 (pGPH11247 in MC4100); (B)
(glpFp18'-lacZ+)1
operon fusion on pJEL250 (pGPH11248 in MC4100). Error bars are as in
Fig. 2.
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These results show that the glpFp18 mutation leading
to a decrease in glpFKX expression is responsible for an
increase in ompF, malT, and MalT-dependent
gene expression during growth in acidic medium. These results confirm
that at least one of the proteins encoded by the glpFKX
operon and expressed at the wild-type level could contribute to the
repression, directly or indirectly, of mal and
ompF gene expression during growth at low pHo.
The amplification of glpK in the cell decreases
mal gene expression.
To further analyze the
involvement of proteins encoded by glpFKX in the repression
of the maltose regulon, the glpFKX copy number was increased
in the wild-type strain by introducing pGPH9925. The
-galactosidase activities of strains harboring either the (malK'-lacZ+)1, the
(malT'-lacZ+)1,
or the
(malP'-lacZ+)1
operon fusion, in the presence of multiple copies of
glpFKX, are shown in Table 2.
At pHo 5 and 7, as GlpF, GlpK, and GlpX were overexpressed
(pGHP9925), malK was fully repressed and
malT and malP expression was reduced by factors
of 3.1 and 2.1.
In order to determine which gene of the glpFKX operon has to
be amplified to exert the repressional effect on the maltose regulon,
internal deletions have been carried out in the glpFKX operon with the glpFKX promoter kept intact (Table 2).
Whatever the pHo, glpF (pGPH9993) or glpX
(pGPH11249) amplification did not reduce maltose gene expression. The
repression of maltose genes was still established in the presence of
GlpF and GlpK when the glpX gene was deleted. The
amplification of glpK alone triggers malK and
malT repression. Thus, GlpK is the protein playing the key
role in the repression observed, even if the strongest repression was
observed when the full glpFKX operon was amplified (full
repression compared to 3.8-fold).
To investigate whether the repression was linked to GlpK itself or to
its kinase activity on glycerol, a promoterless glpFKX operon was cloned under the IPTG
(isopropyl--D-galactopyranoside)-inducible tac promoter of pACT3, resulting in pGPH11542. This
construct allows glpFKX expression without the presence of
glycerol in the growth medium. It was introduced into a strain with the
(malK'-lacZ+)1
operon fusion on the chromosome at att
. Table
3 shows -galactosidase production by
the strain carrying the
(malK'-lacZ+)1
operon fusion with different carbon sources in the presence of IPTG.
Without IPTG added to the growth medium, malK expression was
not affected by the presence of pACT3 or pGPH11542 (data not shown). In
the presence of IPTG, whatever the carbon source (with the exception of
glucose), repression of malK was observed when glpFKX was present (5-fold with glycerol, 2.4-fold with
maltose, 5.1-fold with G3P, 18.4-fold with ribose, 5.9-fold with
succinate, and 2.1-fold with mannose). These data demonstrate that the
maltose regulon repression by GlpK was not specific to growth with
glycerol as the carbon source. Thus, neither phosphorylation of
exogenous glycerol by GlpK nor glycerol metabolism would be required
for repression.
Repression of the maltose regulon required the cAMP-CRP
complex.
The involvement of the cAMP-CRP complex in the repression
of the maltose regulon by a high level of GlpK was characterized using
strains carrying both malTp1 and malTp10
mutations in the control region of malT. These
mutations render malT expression cAMP-CRP independent.
Table 4 shows the effect of
glpFKX amplification on a (malTp1
malTp10'-lacZ+)1
operon fusion and on a
(malK'-lacZ+)1
operon fusion with the malTp1 malTp10 mutations
introduced into the control region of the chromosomal malT
gene. The repression became residual on malK (1.7-fold
instead of full repression) and was cancelled on malT (0.6- instead of 3-fold). The repression observed on malK would be
a consequence of the repression exerted on malT expression.
These data demonstrate that binding of cAMP-CRP to the malT
promoter was required for mal genes to be repressed by
glpFKX amplification.
The use of 
cya crp* derivatives allows the expression of
cAMP-CRP-dependent genes independently of the amount of cAMP present in
the cell. A crp* mutation affects the coding sequence
of crp and enables the Crp* protein to activate
cAMP-CRP-dependent promoters, even in the absence of cAMP. In
the cya crp* background, malK expression was
2-fold repressed instead of fully repressed and malT
expression became 1.2-fold repressed by glpFKX instead of 3-fold (Table 4). The data show that the repression exerted by glpFKX amplification is cancelled when malT
expression is no longer dependent on the amount of cAMP.
Is the repression exerted by glpFKX amplification
restricted to mal genes?
We were interested in
establishing whether the repression observed with the amplification of
glpFKX operates for all pHo-regulated genes. Table
5 shows that glpFKX
amplification lowered ompF transcription twofold while
ompC expression was not significantly modified. These
results agree with an increased ompF expression in
acidic medium in the presence of the glpFp18 mutation
(Fig. 4A). ompA expression, which was previously
reported to be pHo independent, was not repressed by
glpK amplification (Table 5). However, lactose operon expression, which is also known to be pHo independent, was repressed by a factor of 1.8 when glpFKX was
amplified. Thus, the repression exerted by glpFKX
amplification is neither specific to pHo-regulated genes
(lac operon) nor effective on all pHo-regulated loci (ompC gene).
| |
DISCUSSION |
The results presented in this paper demonstrate that
mal and ompF gene expression is correlated with
glpFKX expression levels. A mutation that lowers
glpFKX promoter activity has been isolated. This mutation
allows an increased expression of malT, MalT-dependent genes, and ompF during growth in acidic medium. Conversely,
glpFKX amplification repressed this set of genes. Our
experiments show that GlpK is the protein which is involved,
directly or indirectly, in this repression.
We attempted to discover whether glycerol phosphorylation was required
for establishing this repression. The cloning of an IPTG-inducible
promoter in front of a promoterless glpFKX operon allowed us to overexpress this operon without glycerol or G3P in the growth medium, and this demonstrated that repression of the
maltose system occurred even without exogenous glycerol. This repression was strongest with ribose as the carbon source and no longer
occurred with glucose as the carbon source. Thus, GlpK in excess
exerted its effect independently of glycerol phosphorylation. Conversely, our experiments show that repression of malT
transcription at low pHo was abolished with G3P as the carbon source,
even when glpK expression was induced. These data indicate
that repression at low pHo of malT transcription would
require both glycerol and GlpK.
Eppler and Boos (10) previously analyzed the
glycerol-dependent repression of malT transcription in rich
medium. They demonstrated that glycerol needs to be phosphorylated to
G3P but no further metabolism of G3P is required to establish
repression. Repression is controlled by the level of
IIAGlc phosphorylation and, consequently,
by the cAMP level. In this work, we show that the cAMP-CRP
complex is also involved in the glpFKX
amplification-dependent repression of malT transcription. The cAMP-CRP complex is linked to GlpK via the enzyme
IIAGlc of the PTS. Indeed, glycerol kinase
together with glycerol formed a complex with the unphosphorylated form
of enzyme IIAGlc (25). As the
phosphorylated form of IIAGlc is thought to
stimulate adenylate cyclase (21), we propose that
malT repression by glpFKX amplification could be
mediated by modification of the cAMP level. Overproduction of GlpK in
the cell may titrate the unphosphorylated form of
IIAGlc and avoid its phosphorylation. A smaller
amount of phosphorylated IIAGlc would lead to
weaker adenylate cyclase activation. In addition, the amount of cAMP
would become too limited to fully activate a cAMP-dependent gene, such
as malT. To test this hypothesis, we are currently
investigating the phosphorylation state of IIAGlc
according to the amount of GlpK present and the effect of
glpFKX amplification without IIAGlc in
the cell (crr background).
As the level of mal gene expression was shown to be linked
to the amount of GlpK, we searched for a potential link between an
increased amount of GlpK in acidic medium and maltose gene pHo
regulation. We have shown that glpK expression is modulated by the pHo but with a lower glpFKX promoter activity at low
pHo than at high pHo. Thus, if it exists, the link between the amount of GlpK and maltose gene pHo regulation would be indirect. In acidic
medium, the weaker glpFp18 promoter allowed the alleviation of malT repression that occurs in the parental background.
This result indicates an interplay between malT pHo
regulation and GlpK. This interplay could be at the level of cAMP
synthesis. Thus, we formulated the hypothesis that malT
transcription may follow the cAMP levels in the cell that would, in
turn, change with the pHo. However, a decrease in malT
expression driven by a smaller amount of cAMP-CRP could be demonstrated
only if the amount of complex becomes too small to fully activate the
promoter. As long as there is enough cAMP-CRP to bind to the
low-affinity CRP-binding sites in front of malT, then
malTp would be fully activated. In the wild-type strain,
with glycerol in the growth medium, GlpK is induced and interacts with
IIAGlc, leading to low cAMP synthesis. The effect
of pHo on the amount of cAMP would be observed as pHo regulation of
malT expression. With the glpFp18 mutation, small
amounts of GlpK are produced and more IIAGlc
would be converted to its phosphorylated form, leading to enough cAMP,
whatever the pHo, to fully activate malT transcription. With
maltose as the carbon source, the level of malT
transcription was low regardless of the pHo. This low expression level
could be explained by the same mechanism. Indeed, induced MalK would be
able to interact, as does GlpK, with IIAGlc,
limiting its conversion to the phosphorylated form. With maltose, no
pHo regulation would be seen because the IIAGlc
affinity for MalK is higher than that for GlpK (30) and
the pHo would have no effect on a weakly active adenylate cyclase.
In conclusion, GlpK is not directly responsible for pHo regulation.
However, it may contribute in releasing the pHo effect on adenylate
cyclase through its interaction with IIAGlc,
leading to a level of cAMP below that required for full malT activation. pHo could also influence the phosphorylation state of the
enzyme IIAGlc or the interactions of
IIAGlc with adenylate cyclase by unknown
mechanisms. We are currently investigating these hypotheses.
We thank M. Berlyn, M. Casadaban, C. Cosma, and D. W. Pettigrew for providing bacterial strains and plasmids. We thank Simone Rouzies for excellent technical assistance and wish her a wonderful retirement.
C.C. was supported by a grant from the Ministère en charge de
l'Enseignement Supérieur et de la Recherche. S.A. was a lecturer at University Claude Bernard Lyon 1. This work was supported by a grant
from the Centre National de la Recherche Scientifique (UMR 5122) and
the University Claude Bernard Lyon 1.
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Abstract
Introduction
Present address: INSERM U447, Institut de Biologie de Lille,
Institut Pasteur de Lille, F-59019 Lille Cedex, France.
