Institut de Génétique et
Microbiologie, Université Paris XI, 91405 Orsay Cedex,
France,1 and Institut für
Genetik, Ludwig Maximilians Universität, 80638 Munich,
Germany2
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INTRODUCTION |
LrpC from Bacillus
subtilis belongs to the Lrp/AsnC family of proteins named after
two regulators identified in Escherichia coli: Lrp
(leucine-responsive regulatory protein) and AsnC (3, 6, 17,
27). Lrp from E. coli is a global regulatory protein that controls the expression of about 75 genes (26), whereas AsnC regulates the expression of the asnA gene only, which
codes for asparagine synthetase A (17). Sequences of several
genomes revealed not only the presence of lrp/asnC-like
genes in gram-positive eubacteria and in archaea but also a surprising
multiplicity of these genes in the genome of B. subtilis.
Seven genes encoding proteins of the Lrp/AsnC family, including
lrpC, can be detected in B. subtilis using the
BLAST program (1): lrpC (EMBL accession no.
Z99106), lrpA (yddO) (EMBL accession no. Z99106),
lrpB (yddP) (EMBL accession no. Z99106),
yezC (EMBL accession no. Z99107), ywrC
(EMBL accession no. Z93767), azlB (EMBL accession no.
Y11043), and yugG (EMBL accession no. Z93934). In
addition to the lrpC gene, three other
lrp genes have already been studied: lrpA
(yddO), lrpB (yddP)
(10), and azlB (2). lrpA
and lrpB are implicated in KinB-dependent sporulation,
probably through regulation of glyA transcription by their
corresponding proteins (10). azlB, initially
described as a locus conferring resistance to 4-azaleucine
(44), was found to be a negative regulator of the
azlBCDEF operon involved in branched-chain amino acid
transport (2).
LrpC is a 16.4-kDa protein that cross-reacts with antibodies against
E. coli Lrp (3). It shares 34 and 25% identity
with the E. coli Lrp and AsnC proteins, respectively. An
lrpC mutant does not display a radically altered phenotype,
suggesting that LrpC can be replaced with the other Lrp/AsnC-like
proteins of B. subtilis or that it does not have a
detectable role under laboratory conditions. Furthermore, growth of the
lrpC mutant was not affected. We reported a possible role
for LrpC in entry into sporulation, either by controlling factors that
trigger the onset of sporulation or by regulating early sporulation
genes (3). The LrpC N-terminal region includes a typical
DNA-binding helix-turn-helix motif. Results of heterologous
complementation experiments show that the E. coli ilvIH
operon is repressed by LrpC (3). Taken together, these data
suggest that LrpC is a transcriptional regulator. This idea was tested
by studying the binding of purified LrpC to the promoter of its own
gene and by investigating the regulation of lrpC in vivo.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
An overexpression system
(36) based on T7 RNA polymerase comprised plasmid
pET3blrpC and E. coli strain BL21
(
DE3)pLysS [F
hsdS gal
(cIts857 indI Sam7 nin-5
lacUV5-T7 gene 1) rB
mB pLysS]. Strain 168 (trpC2 BGSC
1A1) was routinely used in this work. The construction of
lrpC mutant strain FC1 (trpC2
lrpC::cat) has been previously described
(3).
Plasmids pUC18 (49), pLysS, pET3b (36), pHP13
(14), and pMUTIN4m (39) have been previously
described. The construction of pT712lrpC, used in sequencing
reactions, has been previously described (3).
pHP13lrpC consists of a 1,184-bp fragment from the B. subtilis 168 chromosome, containing the whole lrpC gene with its transcription-translation signals, cloned between the HindIII and EcoRI sites of pHP13.
For the construction of plasmid pET3blrpC, the
lrpC gene was amplified by PCR using B. subtilis
strain 168 chromosomal DNA as the template and oligonucleotides 1 and 2 (Table 1). These primers were engineered
to contain NdeI and BamHI restriction sites near
the 5' end and also one translation initiation and one termination
codon. The PCR product, 440 bp in length and containing the
lrpC gene, was cleaved by NdeI and
BamHI and then ligated to the
NdeI-BamHI-cleaved pET3b vector, giving the
pET3blrpC plasmid. The lrpC gene was then under
the control of the 
10 promoter of the T7 bacteriophage. The
construction was verified by sequencing.
For the construction of plasmid pUC18prolrpC, the
lrpC promoter region was amplified by PCR using B. subtilis strain 168 chromosomal DNA as the template. Primers 3 and
4 (Table 1) were engineered to contain BamHI restriction
sites near the 5' end. The PCR product, 331 bp in length, was cleaved
by BamHI and ligated to the BamHI-cleaved pUC18
vector, giving the pUC18prolrpC plasmid. The integrity of the cloned region was confirmed by sequencing.
Luria-Bertani (LB) medium was used for routine growth and maintenance
of E. coli cells. LB and glucose minimal Spizizen
(34) media were used to grow B. subtilis
cells in LrpC immunodetection and 
-galactosidase experiments. The
antibiotics used were ampicillin at 50 or 200 µg/ml (for
overexpression experiments), chloramphenicol (CM) at 50 µg/ml, and
erythromycin (ERM) at 5 µg/ml.
Overexpression and purification of the LrpC protein.
Strain
BL21 (DE3)pLysS(pET3blrpC) was grown in LB medium
containing ampicillin at 200 µg/ml and CM at 50 µg/ml to an optical density at 600 nm (OD600) of 0.5. Overexpression was then
induced by 1 mM isopropyl--D-thiogalactopyranoside
(IPTG) for 3 h. Crude extracts prepared from cultures induced for
0, 0.5, 1, 2, and 3 h were fractionated by sodium dodecyl sulfate
(SDS)-16% polyacrylamide gel electrophoresis (PAGE), allowing the
verification of LrpC overproduction.
The purification protocol was finalized by S. Ayora and performed with
modifications as follows. The 16-kDa LrpC protein (predicted molecular
mass, 16,449.86 Da) was purified by monitoring LrpC and E. coli Lrp using immunodetection with antibodies raised against E. coli Lrp. The pellet of a 2-liter culture of strain BL21
(DE3)pLysS(pET3blrpC) induced by IPTG for 3 h and
collected by low-speed centrifugation was resuspended in 50 ml of
buffer A (50 mM Tris-HCl [pH 7.5], 1 mM dithiothreitol [DTT], 1 mM
EDTA, 3 mM phenylmethylsulfonyl fluoride [PMSF], 200 mM NaCl, 5%
glycerol). All further procedures were carried out at 4°C. The cells
were broken in a French press at 25,000 lb/in2. The total
extract was clarified by centrifugation at 3,000 rpm (Sorvall SS34
rotor) for 20 min. Supernatant I was kept. The crude pellet was
sonicated three times for 1 min each time in a Braun Labsonic U/B
apparatus and centrifuged again at 12,000 rpm for 30 min. Supernatant
II was kept. Supernatants I and II were pooled, and polyethylenimine
(10%, pH 7.5) was slowly added with constant stirring to a final
concentration of 0.25%. The solution was centrifuged at 15,000 rpm,
and the pellet was washed several times with buffer A and discarded.
The B. subtilis LrpC protein, which remains in the
supernatant, was precipitated by addition of solid ammonium sulfate to
a final saturation of 60%. The pellet was resuspended in 70% ammonium
sulfate solution and pelleted again.
The protein pellet was resuspended in buffer A, dialyzed against buffer
A containing 100 mM NaCl, and loaded onto a 30-ml phosphocellulose
column equilibrated with buffer A containing 100 mM NaCl. The column
was extensively washed with buffer A containing 100 mM NaCl. The LrpC
protein was eluted from the column by using four steps of 200 mM, 300 mM, 450 mM, and 1 M NaCl in buffer A. E. coli Lrp protein
was eluted at 300 mM NaCl, whereas B. subtilis LrpC was
eluted at 450 mM NaCl. Fractions of the 450 mM NaCl eluate were pooled
and concentrated against Sephadex G200. They were then loaded on a
Sephacryl S100-HR column. The elution fractions corresponding to
proteins with molecular masses of 10 to 70 kDa were monitored by
SDS-PAGE, and those containing pure LrpC were pooled and dialyzed
against buffer A containing 300 mM NaCl. After the last purification
step, the B. subtilis LrpC protein was more than 98% pure
as judged by SDS-PAGE and free of E. coli Lrp as judged by
Western blotting with anti-Lrp antibodies (data not shown). Glycerol
was added to a final concentration of 20%, and samples were stored at
80°C.
The LrpC protein concentration was determined by using a molar
extinction coefficient of 7,860 M1 cm1 at
280 nm.
The isoelectric point (pI) of LrpC (predicted pI, 7.89) was determined
using the PHAST system with isoelectric focusing (IEF) gels 3 to 9 from
Pharmacia under nondenaturing conditions. Pure LrpC (2 µg) was
applied to the middle of the gel, and migration was performed in
accordance with the Pharmacia PHAST IEF protocol. The gel was stained
with Coomassie blue dye, and the pI of LrpC was estimated in accordance
with IEF standards 3 to 9.
Rabbit polyclonal antibodies against LrpC were conventionally prepared
using purified LrpC. Prior to immunodetection, the serum was absorbed
against a crude protein extract of strain FC1 (lrpC).
Curvature of the lrpC promoter region. (i)
Two-dimensional (2D) electrophoresis.
A 200-ng sample of the
331-bp fragment (amplified with primers 3 and 4, corresponding to
fragment d in the curvature assay; Table 1) containing the
lrpC promoter region was mixed with the Gibco BRL 123-bp DNA
ladder. First- and second-dimension electrophoresis was carried out at
4°C with 89 mM Tris borate-2 mM EDTA (pH 8.0) (TBE 1×) in 2%
agarose at 10 V/cm and 8%
acrylamide-N,N'-methylenebisacrylamide (80:1
final ratio) at 2 V/cm, respectively. DNA was stained by incubating the
gel in TBE 1× containing ethidium bromide at 0.2 µg/ml.
(ii) Software analysis.
Curvature of the promoter region was
verified using the DNA ReSCue program (21). This program is
based on the different analysis methods of Koo and Crothers
(18), Bolshoy et al. (4), and de Santis et al.
(11).
(iii) Assay for localization of maximum curvature of the 5'
lrpC region.
Five different 331-bp fragments,
containing the predicted curved region of the lrpC promoter
region at different locations relative to the ends of the fragment (see
Fig. 3A), were amplified by PCR using strain 168 chromosomal DNA as the
template, proofreading Vent DNA polymerase, and the following
combinations of oligonucleotides (Table 1): fragment a, 5 and 6; fragment b, 7 and 8; fragment c, 9 and 10;
fragment d, (this fragment corresponds to the region cloned
in pUC18 and used in 2D analysis and also in retardation experiments),
3 and 4; fragment e, 11 and 12. A 100-ng sample of each
fragment was then subjected to electrophoresis with a 6%
acrylamide-N,N'-methylenebisacrylamide (80:1
final ratio) gel in 40 mM Tris acetate-1 mM EDTA (pH 8.0) at 10 V/cm
and 4°C (see Fig. 3B). For each fragment, the distance migrated, in
centimeters, was plotted against the position of the estimated maximum
curvature relative to the center of the fragment (y x) (see Fig. 3C).
Electrophoretic mobility shift assays.
The different
promoter segments were amplified by PCR using the following
combinations of oligonucleotides (Table 1): 331-bp fragment, 3 and 4 (fragment d of the curvature assay); 265-bp fragment, 13 and
4; 213-bp fragment, 13 and 14; 169-bp fragment, 13 and 12; 128-bp
fragment, 13 and 15; 103-bp fragment, 13 and 16; 90-bp fragment, 5 and
12; 105-bp fragment, 17 and 4; 168-bp fragment, 5 and 4; 130-bp
fragment, 19 and 14.
A typical assay mixture contained 25 mM Tris HCl (pH 8.0), 50 or 150 mM
NaCl, 10% glycerol, 0.1 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol (DTT), 0.1 mM PMSF, 4 mM spermidine, 0.5 nM
32P-end-labeled DNA probe, and various amounts of purified
LrpC protein in a volume of 20 µl. Where indicated, 0.15 µM
competitor DNA (250-fold excess in mass) that corresponds to a
synthetic 266-bp fragment, S15-12, containing tandem repeats of a
15-mer (×10) and flanked by residual pTZ18R polylinker sequences at
both of its ends (47) was added. After incubation for 20 min
at room temperature, the reaction mixture was loaded immediately onto a
6% acrylamide-N,N'-methylenebisacrylamide (80:1
final ratio) gel containing 10% glycerol in 44.5 mM Tris borate-2 mM
EDTA (pH 8.0) (31). Electrophoresis was performed at 10 V/cm
and 4°C. Gels were dried, visualized by autoradiography, and
quantitated with a PhosphorImager (Molecular Dynamics).
Isolation of RNA.
Total RNA was prepared from B. subtilis strains 168pHP13 and 168pHP13lrpC, which
carries lrpC on a five-copy-number plasmid (3).
This strain was used in order to obtain sufficient levels of
lrpC mRNA. Cells were harvested at an OD600 of
1.0 by centrifugation at 4°C. Total RNA was prepared using the Qiagen
RNeasy Kit. The concentration and purity of RNA were determined by
measuring the OD260 and the
OD260/OD280 nm ratio, respectively, and by
analysis on formaldehyde-agarose gels. Forty micrograms of RNA was
precipitated with 3 volumes of ethanol and 1/10 volume of sodium
acetate (3 M, pH 5.2) and resuspended in a final volume of 5 µl of water.
Primer extension.
Forty micrograms of total cell RNA was
mixed with 1 pmol of 5'-end 32P-labeled oligonucleotide
primer (no. 23 in Table 1; positions 515 to 533 in Fig.
1) in 10 µl of avian myeloblastosis
virus (AMV) reverse transcriptase (RT) buffer (50 mM Tris-HCl [pH
8.3], 50 mM KCl, 10 mM MgCl2, 0.5 mM spermidine, 10 mM
DTT). The hybridization conditions were 1 min at 90°C, 10 min at
55°C, and then 15 min on ice. After hybridization, 10 µl of
reaction buffer containing 2 mM each deoxynucleoside triphosphate, 8 mM
DTT, 0.2 volume of 5× AMV RT buffer, RNasin (Promega) at 1 U/µl, and
10 U of AMV RT was added and primer extension was carried out at 42°C
for 1 h. After extension, products were precipitated with 3 volumes of ethanol and 1 M lithium chloride for 30 min at 80°C,
washed with 70% ethanol, and dried. Pellets were resuspended in 12 µl of formamide loading buffer-Tris-EDTA (1:1) and analyzed on a denaturing 6% polyacrylamide gel (Sequagel-6, Prolabo). The same 5'-end 32P-labeled oligonucleotide was used for a sequence
reaction using the Promega fmol DNA sequencing kit and
plasmid pT712lrpC as the template.
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FIG. 1.
Relevant nucleotide sequence of the lrpC 5'
noncoding region. The nucleotide sequence of a 700-bp fragment
containing the whole lrpC 5' noncoding region is presented.
Putative transcriptional and translational signals are underlined. The
15-bp sequence containing 12 bp matching the E. coli Lrp
consensus binding site is shaded. The transcription start site of
P1 (+1), determined by primer extension (see Fig. 7), is
represented by a black arrow. The intrinsically bent segment localized
between promoters P1 and P2 is thickly
underlined. The center of the curvature is indicated by the asterisk.
The gene ydzA, whose function is unknown, and which is
located 185 bp upstream of lrpC, is also indicated, as well
as the first 90 amino acids it encodes. It is transcribed in the
opposite direction with respect to lrpC.
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Detection of LrpC in vivo.
B. subtilis strain 168 was
grown in LB or supplemented Spizizen glucose minimal medium (containing
0.5% glucose, 0.04% MgSO4-7H2O, and 50 µg
of tryptophan per ml). At different growth times, the bacterial
concentration was determined (bacteria were counted on plates of
appropriate dilutions) and samples corresponding to 109
bacteria were harvested. Pellets were washed in buffer M (50 mM Tris
HCl, 1 mM EDTA, 10% glycerol, 250 mM NaCl, 0.1 mM PMSF, 1 mM DTT) and
resuspended in 200 µl of the same buffer. Lysis was carried out by
adding lysozyme at 0.1 mg/ml and incubating the mixture for 10 min at
37°C. A freezing-defreezing procedure was used to obtain complete
cell lysis. DNA was degraded by DNase I at 1 µg/ml in 10 mM
MgCl2 for 10 min at 37°C. Samples were then boiled for 5 min and conserved at 20°C. Protein concentration was determined by
the Bradford assay (31).
A 12-µg sample of each protein extract was fractionated by 0.1%
SDS-16% PAGE (18), blotted onto a nitrocellulose membrane, and incubate with antibodies raised against the LrpC protein. Antibody
binding was visualized using the alkaline phosphatase system. LrpC
protein standards (0.1 to 1 ng of pure protein) were mixed with 12 µg
of crude extracts of lrpC mutant strain FC1 in order to
mimic the strain 168 crude-extract situation and loaded next to protein
samples of strain 168 from LB or glucose minimal medium, and
immunodetection was performed. The established standard curve was then
compared by scanning analysis with LrpC protein detected in crude
extracts from cells grown in LB and glucose minimal medium.
Construction and analysis of lrpC-lacZ fusion.
Plasmid pMUTIN4m (Ermr) (39) contained an
IPTG-inducible promoter, Pspac, followed by a polylinker
sequence for cloning and the lacZ gene preceded by the
strong ribosome-binding site of the spoVG gene. The region
containing the Pspac promoter was deleted from pMUTIN4m,
giving plasmid pFus. Three fragments corresponding to different regions
of the lrpC promoter were amplified by PCR using strain 168 chromosomal DNA as the template, proofreading Vent DNA polymerase, and
the following oligonucleotides (Table 1): fragment 1 (289 bp), 18 and
14; fragment 2 (244 bp), 18 and 12; fragment 3 (205 bp), 18 and 15. Primers were engineered to contain EcoRI or BamHI
restriction sites near the 5' end. The three PCR products were digested
by EcoRI and BamHI and cloned upstream of the
lacZ gene in the EcoRI and BamHI sites
of the pFus plasmid, resulting in plasmids pFus1 to pFus3 (see Fig.
6A). Cloned fragments were sequenced to verify their integrity. These three plasmids were then used independently to transform B. subtilis 168 and FC1 competent cells via a single crossover
(12). Ermr transformants carrying
lacZ transcriptionally fused to lrpC were selected for further analysis and named LF1 to LF3 (168 was the recipient strain) or LF1' to LF3' (FC1 was the recipient strain). Integrations were verified by PCR analysis of the junctions of the
integration (5' junction, primers 20 and 21; 3' junction, primers 22 and 4) (Table 1; see Fig. 6A) and Southern blot analysis performed on
EcoRI-digested chromosomal DNA using the lacZ
gene as the probe. The absence of LrpC protein in LF1', LF2', and LF3' was checked by immunodetection for each recombinant strain (data not shown).
-Galactosidase assays on solid medium were performed as follows. A
drop of each LB culture containing approximately 2 × 106 cells was placed on a petri dish of Spizizen glucose
minimal medium (containing 0.5% glucose, 0.04%
MgSO4-7H2O, and 50 µg of tryptophan per ml)
with added ERM at 5 µg/ml and
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)
at 100 µg/ml. Petri dishes were incubated for 16 h at 37°C and
placed at room temperature until sufficient blue color appeared.
Several transformants of each type were checked for coloration. In
addition, each transformant was plated at least three times to confirm coloration.
-Galactosidase assays in liquid medium were performed as follows.
Strains 168 and LF1 to LF3 were grown at 37°C in Spizizen glucose
minimal medium (containing 0.5% glucose, 0.04%
MgSO4-7H2O, and 50 µg of tryptophan per ml)
and LB with added ERM at 5 µg/ml when necessary. Strains LF1' to LF3'
were grown in the same medium with an additional 5 µg of CM per ml.
Eight milliliters of each culture in the exponential
(OD600, ~0.5) and stationary phases (OD600,
~2) was pelleted, resuspended in 200 µl of lysis solution (100 mM
KHPO4, 20% glycerol, 1 mM DTT, benzamidine at 315 µg/ml, pepstatin A at 1.4 µg/ml, leupeptin at 0.26 µg/ml, and antipain, chymostatin, and PMSF each at 2 µg/ml) and then mechanically broken using glass beads. Lysates were then centrifuged for 20 min at 10,000 × g and 4°C. Ten- and 20-µl samples of each
supernatant were tested in duplicate using the Galacto-Light Assay
protocol (obtained from Tropix) and measured in a luminometer. Relative luminescence units were then expressed per minute, volume of culture (milliliters), and OD unit and converted to -galactosidase units using a standard curve. Arbitrary units therefore correspond to (-galactosidase units/minute/milliliter/OD600 unit) × 106. For average values of two independent
experiments, see Fig. 6C.
Nucleotide sequence accession number.
The complete sequence
of the 5' noncoding region of lrpC has been assigned EMBL
database accession no. Z99106.
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RESULTS |
Purification and properties of LrpC.
The LrpC protein was
overproduced using the T7 RNA polymerase expression system
(36). Briefly, plasmid pET3blrpC, which carries
the lrpC gene downstream of a T7 promoter, was introduced into E. coli strain BL21 (DE3)pLysS and the
lrpC gene was overexpressed following addition of
IPTG. The overproduced 16.4-kDa polypeptide was purified by
conventional column chromatography (see Materials and Methods). The
LrpC polypeptide was free of the 21-kDa E. coli Lrp protein,
as revealed by immunoblotting with antibodies to E. coli Lrp, and was more than 98% pure (data not shown). The LrpC
polypeptide has a calculated pI of 7.89 (Program Compute pI/MW at
http://www.expasy.ch/ch2d/pi_tool.html). The experimentally determined pI of LrpC protein was 7.6 (see Materials and Methods).
LrpC comprises 144 amino acids, corresponding to a molecular mass of
16.4 kDa (deduced from the nucleotide sequence). The molecular mass of
the native LrpC protein, estimated by gel filtration, corresponds to a
tetrameric form of the protein in solution (37).
The lrpC promoter region is intrinsically curved.
Prior to analysis of the DNA-binding properties of the pure LrpC
protein, the upstream region of the lrpC gene was examined for possible regulatory elements. Several interesting features were
observed, and their importance for LrpC binding was assessed. lrpC gene promoter curvature was assessed using the DNA
ReSCue program (see Materials and Methods). Analyses revealed sharply increased curvature around position 363 between the two putative lrpC promoters (Fig. 1 and
2A).
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FIG. 2.
Detection of lrpC 5' noncoding region
curvature. (A) Computer analysis of the curvature of the
lrpC 5' noncoding region. The 700-bp sequence presented in
Fig. 1 was subjected to the DNA ReSCue program (21). Two
curves, determined as described by Koo and Crothers (18)
(black) and Bolshoy et al. (4) (grey), are presented.
Curvature (degrees per base pair) is plotted against position in the
sequence. Maximum curvature is indicated by the arrow. The permuted
fragments used in the curvature assay (see Fig. 3) are aligned under
the 700-bp sequence curvature graph. (B) 2D electrophoresis of a 331-bp
fragment (amplified with primers 3 and 4 and corresponding to fragment
d in the curvature assay) containing the lrpC
promoter region. The 331-bp fragment and the 123-bp linear DNA ladder
(Gibco BRL) were separated in the first dimension in a 2% agarose gel
and in the second dimension in an 8% polyacrylamide gel at 4°C. DNA
was stained by incubating the gel with ethidium bromide at 0.2 µg/ml.
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Curved DNA sequences are common in promoters of prokaryotic genes
(41). The relationship between intrinsic DNA curvature and
transcriptional activity in vivo has been suggested in a number of
cases (15, 24, 35, 48).
The presence of a curved region in the lrpC 5' noncoding
sequence was verified experimentally by analyzing the 331-bp
PCR-amplified lrpC promoter region (positions 157 to 488) by
2D electrophoresis at 4°C (Fig. 2B). The fragment migrated above the
diagonal line formed by the noncurved DNA ladder, as expected for
curved DNA (25).
Next we designed an assay to determine regions of the promoter with
large curve-inducing propensities. According to Wu and Crothers
(46), aberrant migration of curved DNA is more marked the
closer the bend is to the middle of the molecule. Five different fragments of 331 bp in which the putative maximum curvature region (Fig. 3A) was positioned in a distal
(a and e), intermediate (b and
d), or central (c) position of the molecule were
run on a polyacrylamide gel at 4°C and showed different mobilities
(Fig. 3B). A graph representing the distance migrated as a function of
the position of the maximum curvature in relation to the center of the
molecule (y x) revealed that fragment c
is the most retarded fragment, followed by fragments b,
d, a, and e (Fig. 3C). This result is
in good agreement with the localization of the maximum curvature
predicted by in silico analysis. However, fragments a and
e, where the maximum curvature is localized near the end of
the DNA, were also slightly retarded. This could be explained by the
presence of an additional minor curved region(s) that contributes to
the three-dimensional trajectory of the fragments. Furthermore, considering the estimated position of maximum curvature (Fig. 3A),
fragment b should be less retarded than fragment
d. An additional minor curved region, as for fragments
a and e, or alternatively a special conformation
of fragment b undetected by DNA ReSCue analysis could
explain this greater retardation.
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FIG. 3.
Localization of the maximum curvature of the
lrpC 5' noncoding region. (A) Five different ~331-bp
fragments (a to e) of the lrpC 5'
noncoding region were PCR amplified. The positions of the start and end
points of the fragments with respect to the sequence given in Fig. 1
are as follows: a, 302 to 633; b, 251 to 582;
c, 202 to 531; d, 157 to 488; e, 63 to
392. The maximum curvature predicted by in silico analysis (thick
arrow) was displaced along the ~331-bp fragments. x and
y represent the distances, in base pairs, of this putative
curvature maximum from the 5' and 3' extremities of the fragment,
respectively. P1, P2, and ATG of lrpC
are indicated. (B) The five ~331-bp fragments were run on a 6%
polyacrylamide gel at 4°C. Lanes M represent linear 100-bp DNA
ladders (Promega). (C) For each fragment, the distance migrated
(centimeters) was plotted against the position of the predicted
curvature maximum relative to the center of the fragment (y x). Zero on the abscissa corresponds to the position of
curvature maximum predicted by computer analysis.
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DNA-binding characteristics of LrpC.
Computer analysis of the
5' noncoding region of the lrpC gene revealed two putative
promoters (P1 and P2). In addition, a 15-bp
sequence (TTTCATTTTATACTA, coordinates 293 to 307 in Fig. 1)
matching the DNA-binding consensus sequence for the E. coli Lrp protein (12 of 15 positions) overlaps putative promoter
P2 (3, 9).
A 331-bp DNA fragment (0.5 nM) corresponding to the 5' noncoding region
containing P1, the curved region, and P2 was
therefore 32P end labeled and incubated with increasing
concentrations of purified LrpC protein. The formation of protein-DNA
complexes was analyzed by nondenaturing PAGE. As shown in Fig.
4A, LrpC binds its own promoter region in
several steps. At low LrpC concentrations (0.75 to 1.5 nM LrpC in
tetramers), a type I complex was formed, whereas with increasing
protein concentrations (3 to 12 nM), a second, more slowly migrating
complex (type II) accumulated. Both complexes were very distinct and
could correspond to one or two tetramers of LrpC bound to DNA at one or
multiple binding sites. At high LrpC concentrations (24 to 96 nM),
complex II was the major LrpC-DNA complex observed, whereas at even
greater concentrations (192 nM), a higher-order complex that did not
migrate into the gel was observed.
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FIG. 4.
Binding of LrpC to its own promoter region.
(A) The 5' lrpC region (0.5 nM, 331 bp) corresponding to
fragment d (Fig. 3A) was 32P end labeled and
mixed with increasing LrpC concentrations ranging from 0 to 192 nM
(calculated on the basis of the tetramer form of the protein) in a
20-µl final volume. Complexes were resolved on a 6% polyacrylamide
gel. The gel was dried, bands were visualized by autoradiography, and
each band was quantitated with a PhosphorImager (Molecular Dynamics).
The positions of complexes I and II are indicated. (B) Binding isotherm
of free DNA (percent) at various LrpC concentrations. The Kapp value
was estimated as the LrpC concentration required for 50% saturation of
the 331-bp DNA promoter region.
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The bands corresponding to free DNA were quantitated using a
PhosphorImager, and a binding isotherm was then calculated from the
decrease in the level of free DNA. The apparent binding constant (Kapp)
value representing the LrpC concentration required for 50% saturation
of the promoter region (Fig. 4B) is ca. 4 nM at pH 8.0 and room
temperature. The same results were obtained when the experiments were
performed in the presence of a 250-fold excess of nonspecific
competitor DNA. The shape of the curve of LrpC protein binding to the
5' noncoding region indicates cooperative binding of LrpC to DNA; this
was confirmed by the calculation of a Hill coefficient of 1.5 (data not shown).
In certain cases, leucine can modify the binding of E. coli
Lrp to the promoter of a gene it regulates (for example,
ilvIH [30] and gltBDF
[13]). However, binding of LrpC to the 5' noncoding region of lrpC was not modified by addition of
up to 16 mM leucine (data not shown). This result suggests that, in vivo, leucine probably does not affect LrpC binding to its own promoter.
In order to investigate the binding of LrpC to different parts of the
lrpC 5' noncoding region, several DNA fragments were probed
for the ability to bind the pure protein. Ten different DNA
fragments (Fig. 5A) were individually
incubated with 12 nM LrpC (tetrameric) in binding buffer containing
either 50 mM (Fig. 5B) or 150 mM (Fig. 5C) NaCl to discriminate
between high- and low-affinity LrpC binding.
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FIG. 5.
Binding of LrpC to different segments of the
lrpC 5' noncoding region. (A) Ten fragments containing
different parts of the 5' lrpC promoter region were
generated by PCR (see Materials and Methods). Each fragment was
identified by its size in base pairs. Each 32P-end-labeled
fragment (0.5 nM) was mixed with LrpC (12 nM) in a 20-µl final volume
and in the presence of a 250-fold excess in mass of synthetic
competitor S15-12 DNA (0.15 µM). Complexes were formed under two
saline conditions, in 50 mM NaCl (B) and in 150 mM NaCl (C), to
discriminate between high- and low-affinity LrpC binding. Complexes
were resolved in a 6% polyacrylamide gel. The gel was dried, and bands
were visualized by autoradiography and quantitated with a
PhosphorImager (Molecular Dynamics). Percentages of bound DNA in 50 and
150 mM NaCl, respectively, were as follows: 331-bp fragment, 84.6 and
23.7%; 265-bp fragment, 77.5 and 46%; 213-bp fragment, 70.4 and
18.3%; 169-bp fragment, 47 and 6.8%; 128-bp fragment, 36.4 and 4.9%;
103-bp fragment, 14 and 4.9%; 90-bp fragment, 19.8 and 3.5%; 105-bp
fragment, 50.5 and 11.7%; 168-bp fragment, 76.7 and 18.4%.
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LrpC bound with similar affinity to DNA segments of 331 and 265 bp
encompassing P1, P2, and the beginning of the
lrpC gene. When the 265-bp segment was subdivided into three
fragments, 103 bp containing only the P2 region, 90 bp
containing the curved segment, and 105 bp encompassing the
P1 region, a different outcome was observed. At 50 mM NaCl,
the percentages of DNA bound by LrpC were, respectively, 14, ~20, and
~50% for these three fragments (Fig. 5B, lanes 11 and 12, 13 and 14, and 15 and 16), whereas at 150 mM NaCl, these percentages decreased,
respectively, to ~5, 3.5, and ~12% (Fig. 5C, lanes 11 and 12, 13 and 14, and 15 and 16). This indicated that LrpC bound to all three
regions but showed different affinities for them. The highest affinity
was for the P1 region.
In addition, the affinity of LrpC for a 168-bp fragment with
P1 and the curved segment is higher than that for a 169-bp
fragment containing P2 and the curved segment (P1
plus curved segment, ~77% of bound DNA at 50 mM NaCl [Fig. 5B,
lanes 17 and 18] and ~18% at 150 mM [Fig. 5C, lanes 17 and 18];
P2 plus curved segment, ~47% at 50 mM [Fig. 5B, lanes 7 and 8] and ~7% at 150 mM [Fig. 5C, lanes 7 and 8]).
Experiments realized with increasing concentrations of LrpC incubated
independently with the 103-bp (P2), 90-bp (curved
segment), or 105-bp (P1) fragment and the 168-bp
(P1 plus curved segment) or 169-bp (P2 plus
curved segment) fragment confirmed our conclusion that in vitro,
LrpC binds to multiple sites in the 5' noncoding region of its own gene
with preferential affinity for the P1 region (data not shown).
The DNase I cleavage pattern from LrpC-DNA complexes, obtained in
either the presence or the absence of a 250-fold weight excess of
nonspecific competitor DNA, did not reveal any specific protected site
(37). Phosphodiester bonds hypersensitive to DNase I
cleavage, however, were observed in phase with the helical pitch
but were not modified by addition of up to 20 mM leucine (37), confirming that leucine has no influence on the
lrpC promoter-binding activity of LrpC (Fig. 4 and data not
shown). These hypersensitive sites are localized close to the putative
P1 promoter, therefore supporting the hypothesis that LrpC
has the highest affinity for the P1 region (Fig. 5).
However, it is likely that the binding of LrpC to DNA is highly dynamic
and that upon binding to its 5' promoter region, LrpC bends the DNA.
The helical grooves located on the inner surface of the bend are
sterically occluded, while grooves on the outer face are sites of
enhanced DNase I cleavage.
Regulation of lrpC expression.
The fact that LrpC
binds to its own promoter region suggests that LrpC regulates its own
expression, as described for E. coli Lrp (22, 43)
and B. subtilis AzlB (2). To test this
hypothesis, we examined the in vivo expression of lrpC.
Different DNA segments containing either one or both of the putative
promoters, P1 and P2, were fused to a
promoterless lacZ reporter gene carried by plasmid pMUTIN4m
and integrated at the B. subtilis lrpC locus, creating
lacZ transcriptional fusions to lrpC. These
constructions allowed us to follow the expression of the
lrpC gene at its normal locus and to avoid any influence of
chromosome position on gene expression. The constructions also
maintained an intact promoter region controlling the lrpC
gene. They were introduced into both lrpC+ (168 and LF1 to LF3) and lrpC mutant (FC1 and LF1' to LF3')
strains in order to monitor the effect of LrpC on its own expression
(Fig. 6A).
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FIG. 6.
Construction and activities of various
lrpC-lacZ fusions. (A) Three fragments encompassing
different parts of the 5' lrpC region were cloned upstream
of the lacZ gene in plasmid pFus (this work) and sequenced
to verify their integrity. The 15-bp sequence matching the E. coli Lrp consensus binding site is in the black box, whereas the
curved DNA region is in the hatched box. Integrations were then
performed by single crossover in the lrpC locus in a
wild-type (168 leading to LF1 to LF3) or mutant (FC1 leading to LF1' to LF3')
lrpC genetic context. Plasmid DNA is included between the
EcoRI and BamHI restriction sites.
Ermr recombinant strains were used for further analysis.
The lacZ gene is controlled by the P1-P2 region
in LF1 and LF1' and by the P2 region in LF2 and LF2' (with
the whole curved DNA region) and in LF3 and LF3' (with the first third
of the curved DNA region). In LF1, LF2, and LF3, the wild-type
lrpC gene is under the control of the entire
P1-P2 region. Oligonucleotides used for junction
verification and predicted sizes of the corresponding PCR fragments are
indicated. rbs, ribosome-binding site. (B) -Galactosidase
activity of the lrpC-lacZ fusions on solid medium.
Approximately 2 · 106 bacteria of each transformant
type were placed on a petri dish of glucose minimal Spizizen medium
(see Materials and Methods) with ERM at 5 µg/ml and X-Gal at 100 µg/ml. Incubation was performed for 16 h at 37°C and prolonged
at room temperature (25°C) until sufficient blue coloration appeared.
(C) -Galactosidase activity of the lrpC-lacZ fusions in
liquid medium. The different strains were grown in glucose minimal
Spizizen and LB media, and the expression of lrpC was
monitored using the sensitive liquid Galacto-Light Assay protocol
(Tropix). Relative luminescence units were then converted to
-galactosidase units using a standard curve. Arbitrary
-galactosidase units correspond to (-galactosidase units per
minute per milliliter per unit of OD600) × 106.
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Expression of the two putative lrpC
promoters was monitored using both solid (Fig. 6B) and sensitive liquid
(Fig. 6C) -galactosidase assays (see Materials and Methods).
Comparison of reporter gene expression under the control of
P2-P1 (LF1) and P2 alone (LF2 and LF3) clearly
showed that P1 is the major promoter of the lrpC gene in vivo in both rich and minimal media (Fig. 6B and C).
P1 is indeed expressed 10 to 20 times more than
P2 (Fig. 6C). The P2 promoter was found to be
poorly expressed, as revealed by the -galactosidase activity
observed in strains LF2 and LF3, which was only slightly higher than
the background activity (Fig. 6C, strain 168). The predominance of
P1 expression was confirmed by primer extension in which the
transcription start site of P1 was detected whereas that of
P2 was not, even when lrpC was borne on a plasmid
present at five copies per cell (Fig. 7).
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FIG. 7.
Analysis of lrpC promoters by primer
extension. Primer extension of in vivo-produced lrpC mRNA.
Total RNAs from B. subtilis strains 168pHP13 and
168pHP13lrpC were used as templates for primer extension
with 32P-end-labeled primer 23. RNA was prepared from
exponential-phase (OD600 of 1.0) cells. Lanes: 1, control
(without RNA); 2, strain 168pHP13; 3, strain 168pHP13lrpC;
M, markers (Promega); 1 to 3, reaction at 90°C for 1 min, 55°C for
10 min, 0°C for 15 min, and extension at 42°C for 1 h; 4 to 8, control sequence performed with plasmid pT712lrpC as the
template and the same 5'-end 32P-labeled oligonucleotide
primer used for primer extension. The sequence read (corresponding to
positions 411 to 440 in Fig. 1) and that of the complementary strand
are presented. The +1 and 10 sequences of promoter P1 are
indicated. The values to the left are molecular sizes (in bases).
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To investigate the possible autoregulation of lrpC,
expression of the P2-P1-lacZ fusion was measured either in
an lrpC+ context (LF1) or in an lrpC
mutant strain (LF1', lrpC interrupted by the cat
gene). Interruption of lrpC causes not an increase in the
lrpC transcription level but rather a weak consistent
decrease, as shown by (i) the darker coloration of the LF1 strain drop
compared to the LF1' strain drop (Fig. 6B) and (ii) the weaker
expression of lrpC in LF1' compared to LF1 cells grown in LB
or minimal medium (Fig. 6C). Therefore, unlike many lrp/asnC
genes, the lrpC gene is not negatively autoregulated but
appears to be autoregulated in a weak positive manner. This
autoregulation could be due to in vivo binding of LrpC to its own 5'
noncoding region, as seen in vitro (Fig. 4 and 5). The addition of
leucine (100 µg/ml) to the growth medium did not influence
lrpC autoregulation (data not shown). This is in good
agreement with the fact that leucine has no effect on in vitro LrpC
binding to its own promoter.
To obtain an overall view of in vivo lrpC expression, the
production of LrpC was directly monitored by immunodetection under several growth conditions (Fig. 8) (see
Materials and Methods) and the results were compared to those of
-galactosidase assays performed on strain LF1 (Fig. 6C). As shown in
Fig. 8B, the level of LrpC did not change during growth in glucose
minimal medium or even when growth was prolonged over 4 days (data not
shown). The addition of leucine at 100 µg/ml to this medium did not
modify the level of LrpC (data not shown).
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FIG. 8.
Production of lrpC in rich and minimal media
during growth. (A) B. subtilis strain 168 was grown in both
rich LB medium ( ) and glucose minimal supplemented Spizizen medium
(MM;  ) (see Materials and Methods). At different times of growth,
indicated by asterisks, cell concentrations were determined and crude
protein extracts were prepared. (B) A 12-µg sample of each extract
was then subjected to LrpC immunodetection. Lanes: 1, 2.5 ng of pure
LrpC protein; 2 to 5, extracts from glucose minimal medium,
(OD600, 0.2, 0.95, 1.78, and 2.52); 6, protein extract from
FC1 (lrpC mutant) strain; 7 to 10, extracts from LB medium
(OD600, 0.22, 1.13, 2.10, and 3.60).
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Replacement of glucose with Casamino Acids or glycerol did not alter
the LrpC level, although a putative catabolic repression element is
present in the 5' lrpC region (TGGACACGTTTTCA;
7) (coordinates 284 to 297 in Fig. 1). On the
contrary, the production of LrpC appeared to be repressed in
exponentially growing cells in rich LB medium compared to
stationary-phase cells. An estimation of the level of LrpC per cell was
made. In stationary-phase cells in glucose minimal medium and LB
medium, LrpC represents about 0.0058% of the total protein whereas it
decreases to 0.0008% in exponentially growing cells in LB medium.
Whereas this repression in exponential phase represents a 6- to 7-fold
decrease in the LrpC level, measurement of lrpC expression
in LB medium revealed only ~2.5-fold repression (Fig. 6C). This
suggests that an additional factor accounts for the difference in LrpC
production during growth in LB medium (see Discussion). The number of
LrpC molecules per cell was estimated to be about 100 in minimal
medium, 50 in exponential-phase cells in LB medium, and 300 in
stationary-phase cells in LB medium. As expected, the levels of LrpC
are low under the conditions tested and consistent with the weak
lrpC expression levels detected.
| |
DISCUSSION |
Previous studies of the phenotype of an lrpC mutant, as
well as the presence in the LrpC primary sequence of a putative
helix-turn-helix motif, suggested that it functions as a
transcriptional regulator (3, 5). In order to test this
hypothesis, both in vitro DNA-binding properties and in vivo regulatory
functions were assessed. In both cases, we took advantage of the fact
that all of the genes studied that encode proteins from this Lrp/AsnC
family are autoregulated (8, 17, 23, 29, 43).
LrpC binds its own promoter.
We demonstrated that LrpC, like
some other members of the LrpC/AsnC family, binds to the upstream
331-bp lrpC region with an apparent
Kd of about 4 nM (in tetramers) (Fig. 4B). This
Kd is in the range of that measured in the case
of specific binding of E. coli Lrp to various promoters (for
a review, see reference 6). As is the case for
E. coli Lrp, LrpC binding to DNA is specific and
cooperative. In contrast to E. coli Lrp, which is a dimer,
LrpC is a tetramer in solution (37), suggesting that LrpC
binds DNA as a tetramer of four identical subunits.
Analysis of capacities to bind to different parts of the 5'
lrpC region led to the conclusion that the most effective
binding site for LrpC corresponds to a region including the
P1 promoter. However, LrpC is able to bind to multiple sites
in its 5' noncoding region. The 15-bp sequence that resembles the
E. coli Lrp binding site is a minor determinant of LrpC
binding. It is more likely that the DNA structure influences LrpC
binding, since LrpC also binds in vitro to the curved DNA segment
preceding the P1 promoter. This idea has already been
proposed for E. coli Lrp, which seems to bind efficiently
not only to its consensus sequence but also to promoter regions
containing A-T-rich sequences that have been proposed to be
intrinsically curved (43). DNase I protection experiments
performed in parallel with LrpC revealed no specific protected region
but rather hypersensitive sites localized around the P1
region (37), confirming our localization of LrpC binding.
However, the binding of LrpC to multiple sites and the presence of two
different types of complexes (I and II, Fig. 4A) suggest that several
tetramers of LrpC could actually bind this 5' lrpC region.
Indeed, at very high LrpC/DNA ratios, the appearance of higher-order
complexes indicates polymerization and/or aggregation of LrpC on DNA.
Interestingly, the distance separating complexes I and II is more
pronounced for the 331-bp fragment than for the 265- and 213-bp
fragments (Fig. 5B, lanes 1 to 6). This might mean that the length of
the DNA could influence the mode of binding of LrpC or the effect of
LrpC on DNA. Further characterization of LrpC DNA-binding properties
should elucidate this point. It should also determine if and how LrpC
modifies the DNA conformation upon binding, as observed for E. coli Lrp, Crp, Fis, and IHF (16, 38, 43), as well as
the influence of the characteristics of the DNA (length, supercoiling,
etc.) on this modification of the DNA conformation.
Regulation of lrpC expression.
Primer extension
analysis revealed that the P1 promoter is the major promoter
of lrpC in vivo (Fig. 7). Expression from the putative
P2 promoter could not be detected under the conditions tested, suggesting that P2 is very poorly expressed. This
was confirmed by measuring lacZ reporter gene expression
under the control of various parts of the 5' lrpC region
(Fig. 6). However, the computer prediction for this promoter is quite
strong and close to the B. subtilis consensus promoter
region. This suggests that this putative P2 promoter is
expressed conditionally under certain growth conditions not realized in
this work. Monitoring of lrpC expression by the
lacZ reporter gene demonstrated that lrpC is not
negatively autoregulated (Fig. 6), unlike that of many members of the
Lrp/AsnC family. lrpC is weakly positively autoregulated.
This positive autoregulation is not unique to lrpC; several
other B. subtilis regulators are subject to this type of
regulation. For example, the B. subtilis spoIIIG gene
encoding the sporulation sigma factor 
G (32)
or the comK gene, which is essential for the development of
genetic competence (40), are both positively autoregulated. Positive autoregulation of these genes increases the concentration of
the gene products that are required for triggering sporulation or
competence, respectively. In our case, the LrpC protein content in the
cell remains low probably because of the low factor of positive autoregulation.
Since LrpC binds to its promoter region in vitro, it appears that
autoregulation of lrpC is probably due to direct activation by its product. However, we cannot exclude the possibility of an
indirect effect of LrpC, since LrpC could regulate the expression of a
gene coding for a protein that regulates the P1 lrpC promoter.
This weak positive mode of autoregulation is unprecedented in the
Lrp/AsnC family. This difference in the autoregulation mode is in
agreement with our previous indications that LrpC might act in a
different way than E. coli Lrp or other Lrp/AsnC family members. Indeed, B. subtilis LrpC has previously been shown,
using complementation experiments, to repress the E. coli
ilvIH operon normally activated by Lrp (3).
LrpC also differs from Lrp by its oligomeric state since it is
tetrameric whereas Lrp is dimeric (45) and also by its pI
since LrpC is neutral (pI 7.6; our work) whereas Lrp is basic (pI 8.9),
two parameters known for their influence on the mode of DNA binding.
LrpC therefore appears to be an original member of this Lrp/AsnC family
which merits further investigation.
As revealed by monitoring of the expression of lrpC in
strain LF1 (lrpC-lacZ), its transcription is repressed two-
to threefold in the exponential phase compared to the stationary phase
in rich medium (Fig. 6). This repression could be due to various
processes, such as another regulatory protein targeting the 5'
lrpC region. For instance, the 15-bp sequence resembling the
so-called consensus binding sequence of Lrp from E. coli
could be bound by one of the other six B. subtilis Lrp/AsnC
proteins, leading to repression of lrpC. Alternatively, the
intrinsically curved DNA region could be a target for curved
DNA-binding proteins, such as HBsu (50) or the transition
state regulator AbrB (35). It is known that E. coli Lrp is negatively regulated by H-NS, a curved-DNA-binding protein that binds to the lrp promoter region
(28).
Production of LrpC in rich and minimal media.
The production
of the LrpC protein was directly followed during growth in rich and
glucose minimal media. LrpC was expressed at a very low level (ranging
from about 50 to 300 molecules per cell), confirming the weak
-galactosidase activity observed for the lrpC-lacZ
fusions. Furthermore, the level of LrpC was almost constant in cells
growing in glucose minimal medium whereas in LB medium it was reduced
six- to sevenfold during the exponential phase. This reduction could be
explained in part by the repression of lrpC transcription
under the latter conditions. However, the level of this repression is
only two- to threefold, compared to a six- to sevenfold reduction in
LrpC content. This indicates that an additional posttranscriptional
factor accounts for this content reduction, such as less efficient
translation of LrpC mRNA or increased turnover of LrpC protein in the
exponential phase.
Interestingly, azlB, another lrp/asnC-like gene
of B. subtilis, is regulated in a completely opposite way
with respect to lrpC (2). In the wild-type
strain, azlB expression is very low in both rich and minimal
media. In rich medium, weak induction of azlB is observed
during exponential growth. In an azlB mutant, azlB expression is highly derepressed, demonstrating
negative autoregulation, but the induction of expression in the
exponential phase is conserved in both rich and glucose minimal media.
In contrast, for both LrpC and E. coli Lrp, the levels
of proteins are highest during slow growth, pointing to a role for
these proteins under unfavorable growth conditions (20).
Whereas lrpC and azlB are both members of the
B. subtilis lrp-like family, their regulation and function
appear to be radically different.
Possible physiological roles for LrpC.
The amount of LrpC in
the cell corresponds to 10 to 80 tetramers. These levels are not
consistent, at least at first glance, with a global regulatory role for
LrpC such as that observed for E. coli Lrp. This is also the
case for other Lrp-like proteins, such as BkdR, which is present at 20 to 40 tetramers per cell and is only implicated in the regulation of
the branched-chain ketoacid dehydrogenase system in Pseudomonas
putida (23).
Previous experiments have demonstrated the influence of LrpC on
sporulation (3). LrpC is also suspected to play a role in
the regulation of genes required for long-term adaptation to stress.
Indeed, in high-osmolarity minimal medium (1.2 M NaCl), a
lrpC mutant strain grew faster than the wild type during
early exponential growth (C. Beloin, unpublished results). Furthermore, colonies from an lrpC mutant appeared significantly smaller
than those of wild-type cells when grown in LB medium plates at 20°C (Beloin, unpublished). Together, these results suggest that LrpC is a
transcriptional regulator of genes induced during processes requiring
drastic modification of the bacterial physiology.
Several transition state regulators, such as the AbrB, Sin, and Deg
proteins, have been identified in B. subtilis
(33). LrpC could be a part of this complex network
regulating cell responses to environmental changes. A first indication
of this is that the level of LrpC itself responds to environmental
changes. Indeed, the LrpC level in minimal medium (100 molecules per
cell) and in stationary-phase cells in LB medium (300 molecules per
cell) is higher than in exponential-phase cells in LB medium (50 molecules per cell). These data suggest that higher levels of LrpC
correlate with nutrition starvation signals, indicating a role for LrpC in response to starvation.
Although the redundancy of the lrp/asnC genes in B. subtilis and information available for four of these proteins
(LrpA, LrpB, AzlB, and LrpC) favor specialized functions, we believe
they could perhaps act cooperatively in different situations to allow
the cell metabolism to adapt to changes in, e.g., the availability of
nutrients or other parameters of cell growth. Future analysis should
take this possibility into account and should be performed on multiple
mutants to gain insights into a more global role for this family of
regulatory proteins in bacterial physiology.
We thank Eric Larquet and the Laboratory of Cellular and
Molecular Microscopy (IGR, Villejuif, France) for providing the
DNA-ReSCue program and S. Rety for LrpC pI determination. We
thank Y. Hauck, J. Vodolanova, L. Novakova, and A. Blondel for
their technical contribution; M. Bayley for her help with the
English language; S. Joyce for helpful discussions and
corrections; J. L. Gantier for preparing anti-LrpC
antibodies; R. Hasan for help with -galactosidase assays; and H. Putzer for help with RNA extraction.
C. Beloin, R. Exley, and M. Zouine, respectively, acknowledge the
receipt of MENESR (French Ministère de l'Éducation
Nationale, de l'Enseignement Supérieur et de la Recherche) and
FRM (Fondation pour la Recherche Médicale) fellowships, a
European TMR (Training and Mobility of Researchers) Biotechnology grant
(contract BIO4 CT 975141), and MESM (Ministère de l'Enseignement
Supérieur Marocain) and ARC (Association pour la Recherche contre
le Cancer) fellowships. This work was partially supported by grants
from CNRS/Université Paris XI (UMR 2225 and ARC 6794) to F.L.H.
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Abstract
Introduction
Present address: Moyne Institute of Preventive Medicine, Department
of Microbiology, Trinity College, Dublin 2, Republic of Ireland.
