Unité de Régulation de
l'Expression Génétique, Laboratoire de Génomique des
Microorganismes Pathogènes, Institut Pasteur, 75724 Paris
Cedex 15, France,1 and Institut
für Organische Chemie und Biochemie, Fakultät für
Chemie und Pharmazie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg,2 Max-Planck-Institut
für Terrestrische Mikrobiologie, 35043 Marburg,3 and Laboratorium für
Mikrobiologie, Fachbereich Biologie, Philipps-Universität
Marburg, 35032 Marburg,4 Germany
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INTRODUCTION |
Bacillus subtilis was
long considered to be unable to grow in the absence of molecular oxygen
as a terminal electron acceptor. However, as in the case of other
members of the genus Bacillus, the ability of B. subtilis to utilize nitrate as an alternative electron acceptor
has been described by several groups (5, 9, 14, 31). During
the process of anaerobic nitrate ammonification, nitrate is reduced by
a respiratory nitrate reductase (NarGHI) to nitrite, which is
subsequently reduced further to ammonia by a general cellular nitrite
reductase (NasDE) (4, 8, 9, 22). The nar locus,
consisting of the narGHJI operon (encoding respiratory
nitrate reductase), narK (for a potential nitrite extrusion
protein), and the open reading frames ywiC and
ywiD (of unknown function) also contains the regulatory gene
fnr (4). The nitrite reductase genes
nasDE were found downstream of the nasAB operon,
which encodes assimilatory nitrate reductase (24). Two
regulatory systems for the transition from aerobic to anaerobic nitrate
respiratory conditions have already been identified (4, 21).
First, Fnr, a member of Escherichia coli Crp-Fnr regulatory protein family, acts directly on the expression of several anaerobic transcriptional units (i.e., narK and narGHJI),
most likely via interaction with a conserved DNA binding site similar
to the E. coli Crp binding site, which is usually centered
41.5 nucleotides (nt) upstream of the transcriptional start point
(4). Second, ResD-ResE, the pleiotropic two-component
response regulator system encoded by the last two genes of the
resABCDE operon, regulates, directly or indirectly, aerobic
and anaerobic respiration (23, 33). The binding site for the
phosphorylated form of ResD, the active form of the regulator, is still
unknown. B. subtilis fnr is strongly induced in the absence
of oxygen in a Fnr-independent manner (4). This induction is
abolished in a resDE background (23). It is not
known, however, if ResD~P acts directly to activate fnr
transcription or if an unknown intermediary regulator is required. This
contrasts with mainly autonomous fnr function in E. coli (34, 35).
Several groups demonstrated that B. subtilis is able to grow
anaerobically on minimal media in the absence of terminal electron acceptors (8, 19). E. coli and other bacteria use
a mixed acid fermentation for glucose metabolism to form the end
products ethanol, succinate, lactate, acetate, formate, hydrogen, and
carbon dioxide (2). Typical indicators of this process arise
from the activity of its key enzyme, pyruvate formate-lyase (Pfl), which leads to massive excretion of formate and acetate as fermentative by-products. For B. subtilis, Nakano and coworkers
(19) have identified, via nuclear magnetic resonance
analysis, lactate, acetate, acetoin, ethanol, and succinate as main
fermentation products (Fig. 1). No
significant amounts of formate were detected (19), although
the gas phase was not investigated. This observation is supported by
the absence of any obvious counterpart to E. coli Pfl among
the protein sequences deduced from the complete B. subtilis genome sequence (12).
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FIG. 1.
Proposed pathways for anaerobic fermentation and related
catabolism in B. subtilis (modified from references
11 and 19). Enzymes with known
coding genes are as follows: LctE, lactate dehydrogenase; AlsS,
acetolactate synthase; AlsD, acetolactate decarboxylase; Pta,
phosphotransacetylase; Ack, acetate kinase; AcoABC, acetoin
dehydrogenase; Pdh, pyruvate dehydrogenase; PycA, pyruvate carboxylase;
AcsA, acetyl-CoA synthetase. TCA, tricarboxylic acid.
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To study the molecular basis for the coordinated induction for
anaerobic respiration and fermentation, the completely sequenced B. subtilis genome was analyzed for loci potentially
involved in fermentation. Three loci on the B. subtilis
chromosome were investigated for their potential involvement in
fermentative metabolism and corresponding regulation: lctEP,
alsSD, and pta (Fig. 1). The lctE
gene, encoding a protein with similarity to known dissimilatory lactate
dehydrogenases, was identified during the systematic sequencing of the
B. subtilis genome but has not yet been further studied (36). However, 20 years ago, the aerobic regulation of
lactate dehydrogenase formation in B. subtilis was
investigated using biochemical methods (37). The
alsSD operon, which has been shown to encode an acetolactate
synthase and an acetolactate decarboxylase, is responsible for acetoin
production. Its aerobic catabolite-sensitive and growth phase
regulation has been studied (29). Aerobic alsSD transcription is activated in late exponential growth phase. The alsR gene, located upstream of the alsSD operon
and transcribed in the opposite direction, encodes a regulator involved
in this activation process. As observed for alsSD
expression, lactate dehydrogenase synthesis is induced upon the onset
of the stationary phase (37). Finally, acetate formation
from acetyl coenzyme A (acetyl-CoA) is catalyzed in a two-step reaction
by phosphotransacetylase (pta) and acetate kinase
(ack). Two-dimensional gel electrophoresis showed that Pta
formation is aerobically regulated by various stress conditions
(1). The second gene, ack, located in a different position of the genome was found to be subject to catabolite regulation mediated by the catabolite regulator CcpA (6, 27, 32). Here
we describe the investigation of the roles played by lctEP, alsSD, and pta in anaerobic metabolism. Their
oxygen tension- and nitrate-dependent expression dependent on the
regulatory loci resDE and fnr was investigated.
An initial regulatory model is proposed.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
All B. subtilis and E. coli strains used throughout this work
are listed in Table 1. Luria-Bertani
medium was used for standard cultures of B. subtilis and
E. coli if not indicated otherwise (16). For the
investigation of the expression of the various lacZ fusions,
the host strains were grown anaerobically at 37°C on Luria-Bertani
medium supplemented with 20 mM K3PO4 (pH 7), 2 mM (NH4)2SO4, 1 mM
L-glutamic acid, 1 mM L-tryptophan, 0.8 mM L-phenylalanine, 0.005% (wt/vol) ammonium iron(III)
citrate, 1 mM glucose, and, when indicated, 10 mM nitrite or nitrate.
The bacteria were incubated in completely filled flasks with rubber stoppers and with shaking at 100 rpm in an incubation shaker to minimize aggregation of the bacteria. Inoculation was performed aerobically at a 1:100 ratio of aerobically grown overnight culture and
prewarmed medium. Anaerobic conditions were achieved after a short time
through consumption of residual oxygen by the inoculated bacteria.
After five doubling times, in the middle of the exponential growth
phase, samples for 
-galactosidase were taken. The minimal medium for
the high-performance liquid chromatography (HPLC) determination of the
fermentation products produced by the various investigated strains
consisted of 80 mM K2HPO4, 44 mM
KH2PO4, 0.8 mM MgSO4 · 7H2O, 1.5 mM thiamine, 40 µM CaCl2 · 2H2O, 68 µM FeCl2 · 4H2O, 5 µM MnCl2 · 4H2O, 12.5 µM
ZnCl2, 24 µM CuCl2 · 2H2O,
2.5 µM CoCl2 · 6H2O, 2.5 µM
Na2 MoO4 · 2H2O, 50 mM
glucose, 50 mM pyruvate, and where indicated, 10 mM nitrate or 10 mM
nitrite. Anaerobic growth was performed as described above (4, 8,
20).
DNA methods and genetic techniques.
E. coli was
transformed as described by Chung and Miller (3). B. subtilis cells were transformed as described by Kunst and Rapoport
(13). RNA extractions were performed as described by Hagen
and Young (7). Southern blotting and Northern blotting were
performed as described by Sambrook et al. (30). Membranes were further hybridized with nonradioactively digoxigenin-labeled probes (Boehringer, Mannheim, Germany). The lctE- and
lctP-specific DNA fragments used as probes in the Northern
blotting experiments were amplified by PCR using the following pairs of
oligonucleotides: 5'-GCGGAATTCTTTAATCGGAGCGGGT-3' and
5'-ACCTGCGATCCCTCCGC-3' for lctE and
5'-TCTCGAATTCCTTTTGGCTTTAACTGT-3' and
5'-CTCCCGTGACAACCTGC-3' for lctP. Primer
extensions experiments using reverse transcriptase were performed
as described by Pikielny and Rosbash (26). The two
oligonucleotides used as primers for mapping the lctE
promoter were 5'-CGCAAATGCATAACTGCTTCCAAC-3' and
5'-TGACCACAAGCTCATCTGTGATCCC-3'. The SubtiList database was
used to search for sequence patterns in the B. subtilis
genome (17).
Construction of fusion and mutant strains.
B. subtilis
strains containing transcriptional fusions between the E. coli
lacZ gene and the lctE, lctP,
pta, and alsS upstream regions were constructed
using PCR-amplified chromosomal fragments and the integrative plasmid
pJM783 (25) or the pAC5 derivative pDIA5322 (integration at
the amy locus) (Table 1) (15). In pDIA5322, the
pAC5 EcoRI-SacI DNA fragment encompassing the 3' part of the lacZ gene was replaced by the equivalent
fragment including the spoVG initiation codon from pJM783.
The oligonucleotides pairs used and their relative positions with
respect to each start codon are described below. During the
amplification, EcoRI and BamHI restriction sites
were created at opposite ends of the amplified fragment (underlined).
After digestion using these two enzymes, the amplified fragment was
inserted into pJM783 digested with the same enzymes. The plasmid
pDIA5373, containing the lacZ fusion PlctE-lacZ1, was constructed by the integration of the
region 
206 to +434 (with respect to the start codon of
lctE), amplified using the primers
5'-CCGGAATTCTCGGGCTTAAGCGGTTC-3' and
5'-CGCGGATCCAATCACCCGCTCT-3', into pJM783. The
plasmid pDIA5374 harbors the fusion PlctE-lacZ2, consisting
of the region +29 to +434 relative to lctE initiation codon
amplified using the primers
5'-GCGGAATTCTTTAATCGGAGCGGGT-3' and
5'-CGCGGATCCAATCACCCGCTCT-3', inserted into
pJM783. The plasmid pDIA5375, with the fusion
PlctE-lacZ
fnr, was constructed using region 383 to 54
of lctE amplified using the primers
5'-GTAATTGAATTCACCGGATCTTGGCCTGGA-3' and
5'-CCCGGATCCTTTCACATTTATATTGTGCAACACTTCACAAACTTTTGC-3'
and inserted into pJM783 (see Fig. 4). The vector pDIA5376
with PlctP-lacZ contains region +78 to +405 of
lctP amplified using the primers 5'-TCTCGAATTCCTTTTGGCTTTAACTGT-3' and
5'-ATCGGGATCCGAACACCAAAACCGGCC-3' and
integrated into pJM783. Plasmid pDIA5377, containing
PalsS-lacZ1 with region 481 to +394 with respect to the
start codon of alsS amplified using the primers
5'-AGTTGAATTCCTTGTCCGATTTG-3' and 5'-CCGTGGATCCTGCCCTGCTGACGCTAT-3', was
constructed using pJM783. Plasmid pDIA5378 contains the fusion
PalsS-lacZ2 spanning the region +105 to +394 of
alsS, which was amplified using the primers 5'-GCAGGAATTCATGGCCCAAGCAGTC-3' and
5'-CCGTGGATCCTGCCCTGCTGACGCTAT-3'. Plasmid
pDIA5379, harboring PalsS-lacZfnr, was constructed using region 481 to 143 of alsS amplified using the primers
5'-AGTTGAATTCCTTGTCCGATTTG-3' and
5'-GCGAGGATCCGATAAGTTTCACTATACACTC-3' and
integrated into pDIA5322. Pals-lacZfnr was inserted
at the amyE locus of B. subtilis 168 after a
double crossover event to generate strain BSIP1187. PlctE-lacZ1, PlctE-lacZ2,
PlctE-lacZfnr, PlctP-lacZ,
PalsS-lacZ1, and PalsS-lacZ2 were integrated into
the corresponding genes of B. subtilis 168, leading in
some cases to the inactivation of the gene
(PlctE-lacZ2, PlctP-lacZ, and
PalsS-lacZ2). The resulting strains were named BSIP1185,
BSIP1190, BSIP1189, BSIP1191, BSIP1192, and BSIP1173, respectively. The
description of the employed pta-lacZ fusion was given
previously (27). B. subtilis strains in which alsR was disrupted by a spectinomycin resistance gene
(18) were constructed by homologous recombination using
plasmid pDIA5372. pDIA5372 was constructed by the insertion of a
spectinomycin cassette into the unique StuI site of the
pUC18 derivative pDIA5370, previously obtained by a shotgun cloning
experiment (28). In the resulting pDIA5372, the
alsR gene was disrupted 270 bp downstream of the translational start codon. Linearized pDIA5372 was used to
transform B. subtilis 168 to generate BSIP1186.
PlctE-lacZ1 and PalsS-lacZ1 were transferred into
BSIP1186 to generate BSIP1188 and BSIP1194, respectively. The strain
THB361 was constructed by the transfer of
resDE::tet from MH5081 into BSIP1185
via transformation of BSIP1185 using genomic DNA from MH5081 and
appropriate antibiotic selection. All newly created strains were
checked using PCR and hybridization experiments. THB461 and THB561
resulted from the transfer of fnr::spc
(THB2) and narGH::tet (THB1),
respectively, to BSIP1185. BSIP1188 contains
alsR::spec from BSIP1186 in BSIP1185. THB357, THB457, THB557, and BSIP1194 were formed by the transfer of
resDE::tet,
fnr::spc,
narGH::tet, and
alsR::spc from MH5081, THB1, THB2, and
BSIP1186, into BSIP1192, respectively. B. subtilis MMB100
and MMB101 were created by the transformation of BSIP1104 with genomic
DNA prepared from MH5081 and THB2, respectively. BSIP1174 was formed by
the transfer of pta::aphA3 to BSIP1173. MMB110, MMB111, MMB112, and MMB113 were created by the transfer of
narGH::tet or
resDE::tet into THB461 and THB457.
MMB114, MMB115, and MMB116 resulted from the transformation of BSIP1189 with genomic DNA prepared from THB2, MH5081, and THB1, respectively.
HPLC analysis of fermentation products.
Fermentation
products were separated and quantified by HPLC using an LKB-Pharmacia
liquid chromatograph (Amersham Pharmacia Biotech, Freiburg, Germany).
Metabolites were separated on a Eurocat H 300- by 8-mm,
10-µm-pore-size cation-exchange resin (Knauer, Berlin, Germany).
Chromatography was performed at 60°C, with a flow rate of 0.6 ml/min
in 0.01N H2SO4. Eluted compounds were registered and quantified by a refractive index detector equipped with
a computer-powered integrator. Soluble fermentation products were
identified by comparison with retention times and peak areas of
corresponding standards.
-Galactosidase assays.
-Galactosidase activity was
assayed as previously described (16, 20).
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RESULTS AND DISCUSSION |
Analysis of B. subtilis fermentation products using
HPLC.
Previously, Nakano and coworkers demonstrated by nuclear
magnetic resonance the formation of acetate, ethanol, lactate, acetoin, and, under certain conditions, small amounts of 2,3-butanediol from the
carbon source glucose in combination with pyruvate (19). To
monitor more precisely the flow of carbon from glucose and pyruvate
into the various fermentation products, a semiquantitative HPLC
analysis was established. A cation-exchange column was calibrated with
glucose (retention time, 12.1 min), pyruvate (12.8 min), succinate
(16.7 min), lactate (18.3 min), acetate (22.2 min), 2,3-butanediol
(25.8 min), and ethanol (28.1 min). B. subtilis was grown
anaerobically in minimal medium containing 50 mM glucose and 50 mM
pyruvate as carbon sources. Ammonia served as a nitrogen source. Where
indicated, 10 mM nitrate or nitrite was added. Since fermentation
products are excreted by B. subtilis, metabolites present in
the growth medium before and after anaerobic growth were quantified.
Fermentative growth in the presence of 50 mM glucose and 50 mM pyruvate
led to the detection of lactate (38.4 mM), 2,3-butanediol (9.5 mM), and
acetate (13.3 mM). Surprisingly, the addition of the electron acceptors
nitrate and nitrite did not drastically change the overall formation of
fermentation products (Table 2). In both
cases lactate formation was found to be slightly reduced, while the
formation of acetate and 2,3-butanediol was slightly increased. These
observations indicate the presence of the various fermentation
processes during anaerobic respiration in B. subtilis.
In all cases only a minor part of the initial added glucose was
consumed, while the pyruvate was no longer detectable at the end of the
growth (data not shown). At a higher pyruvate concentration (80 mM),
excretion of acetoin was observed (data not shown). Ethanol was not
detected in the growth media under any of the employed conditions using
the HPLC method.
Functional identification of genes involved in anaerobic lactate,
2,3-butanediol, and acetate formation.
Predicted metabolic
pathways for the synthesis of lactate, acetate, and 2,3-butanediol are
shown in Fig. 1 (11, 19). To identify the genetic loci
involved in anaerobic fermentation product formation, we investigated
mutants with mutations in potential fermentation genes previously
identified by the sequencing project or other approaches.
First, the compounds excreted by those mutants when grown under
anaerobic fermentative and respiratory conditions were compared using
HPLC. Lactate is usually produced by reduction of pyruvate in a single
step (Fig. 1). This reaction is catalyzed by lactate dehydrogenase,
with the simultaneous oxidation of one molecule of NADH per molecule of
pyruvate reduced. The lctE gene, potentially encoding
lactate dehydrogenase from B. subtilis, was identified through systematic sequencing (36). In a lctE
mutant (strain BSIP1190), almost no lactate accumulation was observed
under any tested anaerobic condition in minimal (Table 2) or rich (data not shown) medium. The lack of lactate dehydrogenase led to a severe
defect in anaerobic growth in rich and minimal media, although the
lct mutant still accumulates significant amounts of acetate and 2,3-butanediol (Fig. 2; Table 2). In
agreement with the observed lactate accumulation pattern for the
wild-type strain, the observed reduction in anaerobic growth of the
lctE mutant was independent of the presence of alternative
electron acceptors, such as nitrate and nitrite (Fig. 2). These
observations indicate that the lactate dehydrogenase encoded by
lctE is generally important for anaerobic growth. Its
function in the reoxidation of reducing equivalents is not limited to
fermentation but is also important for respiratory growth with nitrate
and nitrite.
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FIG. 2.
Growth of wild-type B. subtilis ( );
mutants with mutations in the alsS ( ), lctE
( ), and pta ( ) genes; and a pta alsS double
mutant ( ) under fermentative conditions (A) and nitrite (B) and
nitrate (C) respiratory conditions. Growth was monitored by
determination of the optical density at 578 nm (OD578 nm)
at the indicated time points. Values reported are the averages from at
least five independent experiments performed in triplicate.
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Aerobic acetoin synthesis from pyruvate in B. subtilis was
studied before (29). It involves two steps catalyzed by
acetolactate synthase and acetolactate decarboxylase, which are encoded
by alsS and alsD, respectively (Fig. 1). The two
genes are organized in an operon (29). An additional step
catalyzed by acetoin reductase converts acetoin to 2,3-butanediol. The
corresponding B. subtilis gene is presently unknown. The
insertional inactivation of the als operon (strain BSIP1173)
totally abolished 2,3-butanediol production under all tested conditions
(Table 2). However, the mutation had only a small effect on the growth
behavior of B. subtilis under the tested anaerobic
conditions (Fig. 2).
The energetically most efficient fermentative pathway is acetate
production. After conversion of pyruvate to acetyl-CoA, the phosphotransacetylase (Pta) and acetate kinase (Ack) form acetate in a
two-step reaction (Fig. 1). Usually, one ATP molecule per molecule of
acetate is produced. Significantly reduced amounts of acetate were
produced in a pta background (BSIP1171) under all tested
conditions (Table 2). A significant reduction of growth was observed
under all tested anaerobic conditions independent of the presence of
alternative electron acceptors. Similar to the conclusions drawn from
the growth behavior of the lctE mutant, pta plays
a general role in the anaerobic energy metabolism. Recent investigation
of the aerobic function of pta revealed its contribution to
the growth in the presence of oxygen (27, 32). The
pta gene is subject to a complex catabolite regulation
involving ccpA, hprK, ptsH1, and
crh (27).
We have also investigated the effect of a pta als double
mutation (BSIP1174). In agreement with the results of the investigation of the strains with single mutations, the double mutant was unable to
produce acetate or 2,3-butanediol and its growth was severely reduced
compared to that of the wild type strains (Table 2; Fig. 2).
Organization of the lctEP operon.
Initial
inspection of the DNA sequence of lctE, encoding lactate
dehydrogenase, and lctP, encoding a putative lactate
permease, suggested the presence of a single transcriptional unit.
Northern blot experiments confirmed this expectation (Fig.
3A). Total cellular RNA was prepared from
wild-type B. subtilis grown under aerobic conditions (Fig.
3A, lanes 1) and under anaerobic conditions in the presence (Fig. 3A,
lanes 2) and absence of nitrate (Fig. 3A, lanes 3). The addition of
fumarate reflects fermentative conditions (Fig. 3A, lanes 4). Probes
specific to lctE and lctP were employed for the
detection of specific mRNA. The lctE probe detected three bands of approximately 2,700, 1,050, and 650 nt. The lctP
probe revealed only the single band of higher molecular weight of
approximately 2,700 nt, which was also seen with the lctE
probe. First, the observed pattern indicated that the two genes are
indeed organized in an operon and that no internal promoter upstream of
lctP was active under the conditions tested. The large
transcript of approximately 2,700 nt has exactly enough coding capacity
for LctE and LctP. The observed transcriptional polarity may contribute
to a lower synthesis of the potential lactate permease compared to
lactate dehydrogenase. Second, comparison of band intensities for the RNAs prepared from B. subtilis grown under various growth
conditions showed that the lctEP operon is significantly
induced on the transcriptional level under anaerobic conditions in the
absence of nitrate (Fig. 3A, lanes 3 and 4). The presence of nitrate
significantly reduced anaerobic lctEP expression. However,
the utilization of increased amounts of total cellular RNA for the
Northern blot and primer extension experiments revealed the presence of
lctEP mRNA even in the presence of nitrate in the growth
medium (data not shown). The molecular basis of the observed regulatory
phenomena was further investigated as outlined below.
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FIG. 3.
mRNA analysis of the lctEP operon. (A)
Northern blot analysis. The lctE- and
lctP-specific probes were generated and the blotting was
performed as outlined in Materials and Methods. (B) Primer extension
mapping. The location of the 5' end of the lctEP mRNA was
deduced from the lengths of the cDNA bands. The length was obtained by
comparison with the sequencing reaction products (in the order A, C, G,
and T) performed with the primer used for the extension reaction. For
both panels, the RNA used in each experiment was extracted from
B. subtilis grown under the following conditions: lane 1, exponential phase in complex medium in the presence of oxygen; lane 2, without oxygen and with 10 mM nitrate; 3, without oxygen or further
additions; 4, without oxygen and with 5 mM fumarate; 5, control without
RNA.
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Promoter structure of the lctEP operon.
The 5' end
of the lctEP mRNA was investigated using the primer
extension technique. As shown in Fig. 3B, the mRNA appeared to start at
a guanosine residue 62 nt upstream of the translational start codon
(Fig. 2B and 3). Potential 10 (CACAAT) and 35
(TTGCAA) regions for a 
A-dependent promoter,
separated by 17 bp, were deduced (Fig.
4). No other mRNA 5' ends were detected
by these mapping experiments, indicating the absence of other active
promoters under the conditions tested. In agreement with the Northern
experiment results and reporter gene fusion experiments (see below),
the strongest primer extension signal was observed using RNA prepared
from fermentatively grown B. subtilis.
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FIG. 4.
DNA sequences of the B. subtilis lctEP and
alsSD promoter regions. The mapped mRNA ends are indicated
by arrows. Predicted 10 and 35 regions are boxed. Predicted Fnr
binding sites are also boxed. The 3' ends of the lctE and
alsS promoter regions in the Plct-lacZFnr and
Pals-lacZFnr fusions are shown. The alsSR
start point was described by Renna et al. (29).
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Analysis of the DNA sequence upstream of the lctEP
operon revealed a palindromic sequence spanning positions 89 to
44 bp upstream of the translational start codon and overlapping the
determined transcriptional start site. Each symmetrical arm contains a
highly conserved potential binding site for the redox regulator Fnr
(Fnr box 1, 5'-TGTGA-AGTGTT-GCACA-3'; Fnr box 2, 5'-TGTGA-AATACT-TCACA-3') (4). The deduced promoter region
is partly within the inverted repeat sequence (Fig. 4). The predicted
10 sequence is relatively poorly conserved and coincides with the
second half of the first potential Fnr site. An initial
characterization of this promoter region is described.
Redox- and nitrate-regulated expression of the lctEP
operon.
In order to further substantiate the
transcriptional regulation highlighted by the mRNA analysis, we have
studied the expression of the lctEP and alsSD
operons by using transcriptional fusions with the E. coli
lacZ gene as a reporter gene. The lacZ fusions were
constructed as described in Materials and Methods. In the strain
carrying PlctE-lacZ1 or PalsS-lacZ1, the
wild-type copy of the corresponding gene was maintained. Integration of
PlctE-lacZ2, PlctP-lacZ, and
PalsS-lacZ2 resulted in the disruption of lctE, lctP, and alsS, respectively. Expression of these
fusions was analyzed during aerobiosis and after a shift to
anaerobiosis in the presence or absence of nitrate or nitrite.
The -galactosidase activity of the PlctE-lacZ1 fusion was
rapidly induced (more than 70-fold) after a shift to anaerobic conditions (Table 3). Anaerobic induction
of the PlctE-lacZ1 fusion was suppressed fivefold if nitrate
was present in the medium. The presence of nitrite had no inhibitory
effect on anaerobic PlctE-lacZ1 expression. The
PlctP-lacZ fusion induction profile was similar to that of
Plct-lacZ1. Inactivation of lctE did not significantly change the observed anaerobic induction of
lctE. However, the nitrate repression of lctE
transcription was reduced. Changes in lctE-lacZ expression
due to structural differences between the employed fusions cannot be
excluded.
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TABLE 3.
Regulation of lctEP expression by oxygen
tension and nitrate, the regulatory loci resDE,
fnr, and alsR, and the nitrate reductase
genes narGHJIa
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Oxygen tension- and nitrate-regulated expression of the
alsSD operon.
Similar to the case for
lctE expression, an approximately 60-fold anaerobic
induction of -galactosidase activity was observed for the
PalsS-lacZ1 fusion in the B. subtilis wild-type
strain grown under fermentative conditions and in the presence of
nitrite (Table 4). Again, nitrate reduced
alsS expression threefold. Mutation of alsS did
not influence anaerobic PalsS-lacZ2 expression (Table 4). To
investigate the participation of various known redox regulators and of
AlsR in redox- and nitrate-dependent alsSD and
lctEP expression, lacZ fusions were tested in
B. subtilis strains carrying mutations in the
fnr, resDE, and alsR loci.
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TABLE 4.
Regulation of als expression by oxygen tension
and nitrate, the regulatory loci resDE, fnr, and
alsR, and the nitrate reductase
genes narGHJIa
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Participation of fnr, resDE, and
alsR in lctEP and alsSD
expression.
Mutation of fnr reduced anaerobic
PlctE-lacZ1 and PalsS-lacZ1 expression by half
(Tables 3 and 4). Expression of lctEP and alsSD
is subject to arfM (ywiD) regulation
(10; M. Marino, H. Cruz Ramos, T. Hoffman, P. Glaser, and D. Jahn, unpublished observations). Transcription of
arfM (ywiD) was found to be completely
fnr dependent. Due to the similar degrees of arfM
and fnr regulation, it was concluded that there is an
indirect participation of fnr in lctEP and
alsSD expression via arfM induction
(10; M. Marino et al., unpublished
observations). Moreover, the location and initial analysis of the
potential Fnr binding sites (see below) made their participation in a
direct Fnr-mediated anaerobic induction of both operons very unlikely.
The second consequence of fnr mutation was the complete loss
of nitrate repression on both operons (Tables 3 and 4).
Interestingly, mutation of resDE also led to the loss of
nitrate repression. These findings prompted us to investigate the
participation of narGHJI, encoding respiratory nitrate
reductase, in nitrate repression, since narGHJI expression
is strictly dependent on fnr and resDE. As shown
in Tables 3 and 4, narGH deletion also completely abolished nitrate repression of both operons. These results suggest that the observed nitrate regulation depends on the enzymatic nitrate reductase activity. Consequently, only an indirect participation of
fnr and resDE via narGHJI induction in
nitrate repression of anaerobic lctEP and alsSD
transcription is suggested.
A clear difference was observed for the consequences of a
resDE mutation on the overall anaerobic lctEP and
alsSD expression. While PlctE-lacZ1 expression
was reduced three- to fourfold in a resDE mutant,
PalsS-lacZ1 expression remained mainly unchanged. This
observation indicated an additional direct or indirect participation of
resDE in anaerobic lctEP induction.
In agreement with the results obtained for the investigation of
lctEP expression in single fnr and
resDE mutants, analysis of a PlctE-lacZ1 fusion
in an fnr resDE double mutant revealed significantly reduced
anaerobic induction with the complete loss of nitrate repression (Table
3). An fnr resDE double mutant led to the reduction of
alsSD expression slightly below the level determined for the
fnr mutant alone and to the complete loss of nitrate
repression. This is again in agreement with the analysis of single
regulatory mutants, where the redox regulation of alsSD did
not significantly respond to resDE mutation (Table 4). In agreement with the observed effects of single fnr and
narGH mutation on lctEP and alsSD
expression, an fnr narGH double mutation reduced anaerobic
lctEP and alsSD induction and completely
abolished nitrate repression (Table 3).
The regulatory gene alsR, located upstream of the
alsSD operon, was found to be essential for both
PlctE-lacZ and PalsS-lacZ expression under all
tested anaerobic conditions. Previous investigations suggested the
intracellular pH as well as the acetate concentration in the growth
medium as possible signals for AlsR-dependent regulation (29).
Initial analysis of the potential Fnr binding site in the
lctEP and alsSD promoters.
The analysis of
the lct promoter region revealed a complex structure. In
order to assess the role of the apparent Fnr binding sites in
regulation, a truncated fusion was constructed
(PlctE-lacZfnr [Fig. 4]). In this fusion only the first
five bases after the transcription start point were kept, while the
downstream half of the second putative Fnr site was removed as shown in
Fig. 4. The expression of this PlctE-lacZfnr fusion was
monitored under aerobic and various anaerobic growth conditions (Table
3). The loss of the second half site led to a higher anaerobic
induction of lctE expression and a significant decrease of
nitrate repression. These results indicate that this region might be
involved in a mechanism of repression the lct transcription.
In agreement with the data obtained for the analysis of the wild-type
lctE promoter, resDE mutation significantly
reduced anaerobic lctEP induction via the mutated promoter.
However, the values obtained for the resDE mutant still
documented the same degree of derepression observed for the wild-type
strain (Table 3). Moreover, similar to the wild-type lctEP
promoter, the mutated promoter revealed significantly reduced nitrate
repression in a resDE mutant. In complete agreement with the
case for the wild-type lctEP promoter, the mutated promoter
responded to a narGH mutation with complete loss of nitrate
repression. The degree of anaerobic depression of the mutated promoter
in the narGH mutant was identical to that of the wild-type
strain. Surprisingly, mutation of fnr did not decrease
transcription from the mutated lctEP promoter, while the
native promoter partially lost its anaerobic induction capacity. This
observation indicated the importance of the mutated promoter sequence
for direct or indirect influence of fnr on lctEP expression.
Interestingly, a similar Fnr-like binding site is found in the vicinity
of the als operon transcription starting point (Fig. 4). In order to assess if this sequence is involved in als
regulation, the Pals-lacZfnr fusion was constructed. In
this fusion only the first three bases downstream of the transcription
starting point were kept (Fig. 4). In the resulting strain, BSIP1187,
the transcription of the Pals-lacZfnr fusion was
abolished during both aerobiosis and anaerobiosis (Table 4). These
results could be interpreted in two ways: either the binding of an
essential activator has been abolished or the integrity of the overall
promoter structure has been disturbed in this construct. In both cases the effect of an fnr gene mutation on lctEP and
alsSD expression contrasts with the results of the
deleted-promoter study. In the case of lctEP, an
fnr mutation decreased lctEP transcription, while
the mutation of the putative Fnr box derepressed lctEP
transcription. The location of the putative Fnr box with respect to the
transcriptional start site of alsSD suggested that a protein
binding to this site would act as a repressor. However, an
fnr mutation again led to decreased transcription, and
deletion of the putative Fnr box totally abolished transcription.
These results indicate that the Fnr box-like elements of the
lctEP and alsSD promoters do not serve known
Fnr-dependent regulatory functions.
Expression of pta under anaerobic conditions.
The
third fermentative locus, pta, was investigated for its
potential oxygen- or nitrate-dependent control of transcription (Table
5). In agreement with its general aerobic
and anaerobic function, no obvious regulation by oxygen tension,
nitrate, or nitrite was observed. Mutation of resDE and
fnr did not change pta transcription. These data
in combination with the observed pta growth phenotypes and a
recent analysis of anaerobic pta function (27)
demonstrated the overall importance of acetate formation for aerobic
and anaerobic growth.
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|
TABLE 5.
Investigation of dependence of pta expression
on changing oxygen tension and nitrate as well
as nitritea
|
|
A model for the regulation of anaerobic fermentation genes in
B. subtilis.
The lctEP and alsSD
operons are an integral part of the anaerobic modulon of
B. subtilis. Under anaerobic conditions, both are induced
via a regulatory cascade from an unknown sensor via ResDE, Fnr, and
ArfM (Fig. 5). However, ArfM activation
is responsible for only a part of the observed degree of anaerobic
lctEP and alsSD induction. Additional,
yet-unknown, redox regulatory components are required for full
anaerobic gene expression. A similar regulatory cascade was recently
determined for B. subtilis hemN and hemZ transcription (10).
Nitrate only partially represses lctEP and alsSD
expression. The nitrate regulatory system involved is still unknown.
Intact nitrate reductase and lactate dehydrogenase are required to
allow nitrate regulation. One could speculate that the redox status of
the cell (NAD-to-NADH ratio) or nitrate-dependent membrane-localized electron flow is a signal for this unknown system. Alternatively, an
already-known regulatory system responding to the outlined changes in
intracellular parameters could indirectly mediate nitrate regulation in
B. subtilis.
Lactate and 2,3-butanediol formation was clearly detectable in the
presence of nitrate and nitrite, indicating the importance of NADH
reoxidation even for anaerobic respiratory growth. The obvious absence
of a proton translocating NADH dehydrogenase of the Nuo type from the
B. subtilis genome and the observed strictly aerobic
expression of the non-proton-pumping NADH dehydrogenase of the Ndh type
explain the observed general anaerobic lctEP and alsSD expression (13, 17; Marino et al.,
unpublished observations).
Both operons are additionally subject to AlsR regulation. The
postulated signal(s) for AlsR activity is the intracellular pH and/or
acetate or derived metabolites (29). The observed rapid
anaerobic induction of alsSD and lctEP could
result from the accumulation of acidic compounds like pyruvate and
acetate. Further experiments should shed light on the complex
regulatory systems involved in the control of anaerobic metabolism of
B. subtilis.
Finally, acetate formation is a general part of the aerobic and
anaerobic B. subtilis metabolism (27). In
agreement with this observation, pta expression is not
subject to redox regulation.
This work was supported by grants from the Deutsche
Forschungsgemeinschaft, the Max-Planck-Gesellschaft, the
Sonderforschungsbereich 388, Fonds der Chemischen Industrie, and the
Graduiertenkolleg "Biochemie der Enzyme." E.-P.-S. is a fellow of
the European Union Biotech Programme (contract ERBB102 CT930272). This
research was also supported by grants from the Ministère de
l'Education Nationale de la Recherche et de la Technologie, Centre
National de la Recherche Scientifique (URA1129), Institut National de
la Recherche Agronomique, Institut Pasteur, Université
Paris 7, and European Union Biotech Programme (contracts ERBB102
CT930272 and ERBB104 CT960655).
We are indebted to M. Nakano (Louisiana State University) and F. M. Hulett (University of Illinois at Chicago) for the gift of B. subtilis mutants. We thank R. K. Thauer
(Max-Planck-Institute, Marburg, Germany) for continuous support.
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Abstract
Introduction
Present address: Department of Molecular Biology and Biotechnology,
The University of Sheffield, Sheffield S10 2TN, Great Britain.
