Journal of Bacteriology, December 2001, p. 6815-6821, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6815-6821.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Modulation of Anaerobic Energy Metabolism of
Bacillus subtilis by arfM
(ywiD)
Marco
Marino,1,2
Hugo Cruz
Ramos,3,
Tamara
Hoffmann,1,4
Philippe
Glaser,3 and
Dieter
Jahn1,2,*
Institut für Organische Chemie und
Biochemie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg,1 Institut für
Mikrobiologie, Technische Universität Braunschweig, 38106 Braunschweig,2 and Max-Planck-Institut
für Terrestrische Mikrobiologie, 35043 Marburg, and
Laboratorium für Mikrobiologie, Fachbereich Biologie,
Philipps-Universität Marburg, 35032 Marburg,4 Germany, and Unité de
Régulation de l'Expression Génétique, Institut
Pasteur, 75724 Paris Cedex 15, France3
Received 23 April 2001/Accepted 23 August 2001
 |
ABSTRACT |
Bacillus subtilis grows under anaerobic conditions
utilizing nitrate ammonification and various fermentative processes.
The two-component regulatory system ResDE and the redox regulator Fnr
are the currently known parts of the regulatory system for anaerobic
adaptation. Mutation of the open reading frame ywiD located upstream of the respiratory nitrate reductase operon
narGHJI resulted in elimination of the contribution of
nitrite dissimilation to anaerobic nitrate respiratory growth.
Significantly reduced nitrite reductase (NasDE) activity was detected,
while respiratory nitrate reductase activity was unchanged. Anaerobic
induction of nasDE expression was found to be
significantly dependent on intact ywiD, while anaerobic
narGHJI expression was ywiD independent. Anaerobic transcription of hmp, encoding a
flavohemoglobin-like protein, and of the fermentative operons
lctEP and alsSD, responsible for lactate
and acetoin formation, was partially dependent on ywiD.
Expression of pta, encoding phosphotransacetylase
involved in fermentative acetate formation, was not influenced by
ywiD. Transcription of the ywiD gene was
anaerobically induced by the redox regulator Fnr via the conserved
Fnr-box (TGTGA-6N-TCACT) centered 40.5 bp upstream of the
transcriptional start site. Anaerobic induction of ywiD
by resDE was found to be indirect via
resDE-dependent activation of fnr. The
ywiD gene is subject to autorepression and nitrite
repression. These results suggest a ResDE 
Fnr YwiD regulatory
cascade for the modulation of genes involved in the anaerobic
metabolism of B. subtilis. Therefore,
ywiD was renamed arfM for anaerobic
respiration and fermentation modulator.
| |
INTRODUCTION |
Under anaerobic growth conditions,
Bacillus subtilis can generate ATP via nitrate
ammonification or fermentation (2, 5, 14). During nitrate
respiration, nitrate is reduced by the respiratory nitrate reductase
(NarGHI) to nitrite (2, 5, 9). Nitrite is further reduced
to ammonia by a general nitrite reductase (NasDE) (4, 13).
The latter enzyme also contributes to the nitrite assimilation process
(13). During anaerobic fermentation, carbon sources are
transformed via pyruvate into the end products lactate, acetoin,
2,3-butanediol, ethanol, acetate, and succinate (3, 12).
NAD+ regeneration is primarily mediated by a
cytoplasmic lactate dehydrogenase, encoded by lctE, that
converts pyruvate to lactate (3). Acetoin is synthesized
from pyruvate in a two-step reaction catalyzed by acetolactate synthase
and acetolactate decarboxylase, encoded by the alsSD operon
(3, 20). Subsequently, acetoin is converted to
2,3-butanediol by acetoin reductase (12). The third major fermentation product, acetate, is formed from acetyl-coenzyme A in a
two-step reaction catalyzed by phosphotransacetylase and acetate
kinase, encoded by pta and ack, respectively. The
latter step usually leads to the formation of ATP.
Due to the drastically different ATP yields of respiratory and
fermentative processes, bacteria usually use a fine-tuned regulatory system to maintain the most efficient mode of ATP generation. In
B. subtilis only parts of the anaerobic redox regulatory
system are known. The pleiotropic two-component regulating system
ResDE, encoded by the resABCDE operon, is activated by an
unknown redox-sensing system (21). Activated ResD binds
directly to DNA elements (TTTGTGAAT) located within anaerobically
induced promoter regions. Activator binding at this conserved promoter
element and transcriptional activation were demonstrated for
nasDE, the flavohemoglobin gene hmp, and the
redox regulatory gene fnr (14, 16, 21). The redox regulator Fnr, possibly containing an iron sulfur cluster similar
to its Escherichia coli counterpart, is subsequently
responsible for the induction of the narGHJI operon and
narK, encoding respiratory nitrate reductase and a potential
nitrite extrusion protein, respectively (2).
All known Fnr-regulated genes have a highly conserved potential
B. subtilis Fnr-binding site
(TGTGA-N6-TCACA) in their promoter regions.
Additional potential Fnr-binding sites were found in the 5' regions of
a second potential nitrate/nitrite transporter gene, ywcJ,
the fermentation operons lctEP and alsSD, and
ywiD, encoding a protein of unknown function (2,
3).
The regulation of genes involved in fermentation was described recently
(3). Transcription of alsSD and
lctEP is induced anaerobically and repressed by the presence
of nitrate (3). However, Fnr is only partially responsible
for anaerobic lctEP and alsSD induction. These
findings, in combination with the incompletely understood molecular
basis for anaerobic nasDE and hmp induction, raise the possibility of additional redox regulatory components in
B. subtilis. Here we provide evidence that ywiD,
located in the 5' region of the narGHJI operon, is an
important part of the anaerobic regulatory system, responsible for the
modulation of anaerobic gene expression. Since ywiD
expression is Fnr dependent, a regulatory cascade from an unknown
sensor proceeding via resDE through fnr and
ywiD to multiple target genes is proposed.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
All B. subtilis strains used are listed in Table
1. 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 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 (4, 13).
Inoculation of the test culture was performed under the described
conditions and started in all experiments with identical amounts of
cultured cells. For all strains tested, 
-galactosidase activities
were followed over the whole growth phase. Values obtained from
comparable growth phases are listed. For -galactosidase assays,
cells were harvested at an appropriate optical density at 578 nm
(OD578) by centrifugation. The cell pellet was
resuspended in 400 µl of Z-buffer (60 mM
Na2HPO4 · 2H2O, 40 mM
NaH2PO4 · H2O, 10 mM KCl, 1 mM
MgSO4 · 7H2O, 50 mM
-mercaptoethanol, pH 7.0). Lysozyme-DNase solution (2 µl; 8 mg of
lysozyme in 950 µl of sterile water and 50 µl of DNase [2.5 mg/ml
in 3 M sodium acetate]) was added and incubated for 15 min at 37°C.
The crude cell extract was centrifuged for 5 min to remove cell debris.
The supernatant was recovered. Protein concentration of the cell
extract was determined using the Roti-Quant (Roth, Karlsruhe, Germany)
protein detection assay. Then 600 µl of Z-buffer was added to 200 µl of crude cell extract.
The reaction was started by adding 200 µl of
ortho-nitrophenylgalactopyranoside stock solution (4 mg/ml).
To stop the reaction, 500 µl of 1 M
Na2CO3 was added, the
reaction time was noted, and the OD420 was
determined versus the reference reaction. The specific activity was
determined using the following equation: units per milligram of
protein = 1,500/[test volume (milliliters) × time (minutes) × protein concentration (milligrams per milliliter)] × OD420.
For the growth experiments, minimal medium containing 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
Na2MoO4 · 2H2O, 50 mM glucose, and, where indicated, 10 mM
nitrate or 10 mM nitrite was used. Antibiotics were added when
necessary at the following concentrations (milligrams per liter):
ampicillin, 100; chloramphenicol, 5; kanamycin, 10; and spectinomycin,
60. Bacteria were grown at 37°C in all experiments.
DNA manipulations and genetic techniques.
E. coli
was transformed as described by Chung and Miller (1).
B. subtilis cells were transformed as described before
(7). Transcriptional fusions with the E. coli
lacZ gene were constructed using integrative plasmid pJM783
(16). A pUC18 derivative was obtained during the pDIA5348
shotgun cloning experiment described before (2),
encompassing the complete intergenic region between the ywiC
and ywiD genes.
To construct a transcriptional fusion with the ywiD gene,
the chromosomal insert from the pUC18 derivative was excised as a
~0.34-kb HaeIII-BclI fragment and inserted
between the SmaI and BamHI sites in pJM783,
leading to plasmid pDIA5562. The lacZ gene in this construct
was placed after the 38th codon of the ywiD gene. Both
transcriptional fusions were introduced into the B. subtilis
168 chromosome by Campbell-type recombination events to generate
B. subtilis BSIP1203 and BSIP1204, respectively. The ywiD-lacZ fusion was transferred from BSIP1204
into the B. subtilis JH642-based fnr mutant THB2,
the resDE mutant MH5081, the resDE mutant LAB2313
carrying fnr under the control of the IPTG
(isopropylthiogalactopyranoside)-inducible Pspac promoter,
and the ywiD mutant to generate B. subtilis MMB2, MMB4, MMB20, and MMB21, respectively.
A B. subtilis strain in which ywiD was
interrupted by a kanamycin resistance gene (11) was
constructed by homologous recombination using pDIA5564. Plasmid
pDIA5564 was created from another pUC18 derivative (2),
encompassing the 5' end of the ywiC gene and the complete
ywiD gene (pDG782). The plasmid was linearized by BclI and ligated to a BamHI-BglII
restriction site-flanked kanamycin cassette, interrupting the
ywiD gene after the 38th codon. Plasmid pDIA5564 was
linearized and used to transform B. subtilis JH642.
A strain in which the wild-type ywiD gene was replaced by
the disrupted copy, ywiD::kan (THB110),
was selected as a kanamycin-resistant transformant. The
ywiD::kan mutation was transferred into
LAB2854 (nasD-lacZ), LAB2143
(narG-lacZ), MMB61
(lctE-lacZ), MMB57
(alsS-lacZ), MMB101
(pta-lacZ), and LAB2000
(hmp-lacZ) to generate MMB10, MMB9, MMB15, MMB14,
MMB102, and MMB8, respectively. The ywiD mutation of THB110
was transferred into the fnr mutant THB2 and the
resDE mutant MH5081 to generate the ywiD fnr and
ywiD resDE double mutants MMB40 and MMB41, respectively.
Finally, the nasD-lacZ (from LAB2854), narG-lacZ (LAB2143),
lctE-lacZ (BSIP1185),
alsS-lacZ (BSIP1192), ywiD-lacZ (BSIP1204), and
hmp-lacZ (LAB2000) fusions were transferred to
the double mutants, resulting in the strains listed in Table 1. We
failed to obtain a strain carrying an alsS-lacZ
fusion in a ywiD fnr mutant background.
Mutation of the fnr site upstream of ywiD from
5'-TGTGA-AATACA-TCACT-3' to
5'-CCTGA-AATACA-TCACT-3' localized on the
ywiD-lacZ fusion-carrying plasmid pDIA5562 was
performed using the Quick Change site-directed mutagenesis kit
(Stratagene, Heidelberg, Germany) according to the instructions of the
manufacturer. The resulting plasmid, pDIA5562
Fnr, was introduced
into JH642 to generate MMB25 (ywiD
fnr-lacZ).
PCR and Southern blotting experiments were used to confirm the
appropriate substitution of the wild-type gene by the mutated copy in
mutant strains and to verify that only a single copy of the
lacZ fusion was integrated.
Primer extension analysis of 5' end of ywiD
mRNA.
Total cellular RNA was prepared from B. subtilis
using the RnEasy minikit (Qiagen, Hilden, Germany), according to the
instructions of the manufacturer. The 5' ends of mRNAs encoded by the
ywiD gene were mapped with an oligonucleotide that was
complementary to positions 55 to 78 (5'-GTGCCTCTATCCATTGTCGAAACC-3') of the ywiD gene
and fluorescence labeled with 5'-indodicarbocyanine (5'-Cy5). For each
experiment, 20 to 100 µg of RNA was incubated with 0.2 pmol of
labeled primer for 3 min at 70°C in 34 mM Tris-HCl (pH 8.3)-50 mM
NaCl-5 mM MgCl2-5 mM dithiothreitol. The
primer-RNA hybrids were extended with 10 U of avian myeloblastosis
virus reverse transcriptase and 0.5 mM each nucleoside triphosphate, 12.5 mM dithiothreitol, 12.5 mM Tris-HCl (pH 8.3), and 7.5 mM MgCl2 for 1 h at 42°C in the presence of
10 U of RNasin. Extension products were purified by phenol extraction,
subjected to denaturing polyacrylamide gel electrophoresis (PAGE), and
monitored by the ALF Express system (Amersham Pharmacia Biotech,
Freiburg, Germany). A sequencing reaction performed with the same
primer set was run in parallel on the same gel and allowed direct
identification of the ywiD mRNA 5' end.
HPLC analysis of B. subtilis fermentation
products.
Analysis of excreted fermentation product by
high-pressure liquid chromatography was performed as outlined before
(3).
| |
RESULTS AND DISCUSSION |
Reduced anaerobic growth of B. subtilis
ywiD mutant.
In the 5' region of the
narGHJI operon encoding respiratory nitrate reductase, an
open reading frame of unknown function termed ywiD was found
(Fig. 1). The open reading frame
ywiD would encode a protein of 158 amino acid residues and a
calculated molecular mass of 18,137 Da. The deduced protein showed no
significant similarity to any other protein of known function in the
database. To investigate its potential participation in anaerobic
growth processes, a genomic knockout mutation of the gene was
constructed and its growth behavior in minimal medium was compared to
that of wild-type B. subtilis under aerobic and various
anaerobic growth conditions.
View larger version (12K):
[in this window]
[in a new window]
|
FIG. 1.
nar locus of B. subtilis.
The fnr gene encodes a redox regulatory protein,
narK a putative nitrate/nitrite transporter protein, and
narGHJI the respiratory nitrate reductase. The open
reading frame arfM is the subject of this investigation.
Potential Fnr binding sites are boxed. The 5' end of the
arfM mRNA is indicated by the arrow.
|
|
Deletion of ywiD had no obvious influence on aerobic growth
(Fig. 2). However, anaerobic growth in
minimal medium was significantly reduced in the presence of nitrate or
nitrite and under fermentative conditions (Fig. 2). The typical
biphasic anaerobic growth curve for wild-type B. subtilis on
minimal medium in the presence of nitrate was not observed for the
ywiD mutant. The biphasic character of the curve for
anaerobic nitrate respiratory growth results from the sequential
utilization of first nitrate and then nitrite (4). The
observed growth curve of the ywiD mutant mirrored the
anaerobic growth behavior of a nasD mutant on
nitrate-containing medium, missing the growth enhancement from nitrite
dissimilation via nasDE-encoded nitrite reductase
(13). In agreement with this observation, reduced
anaerobic growth of the ywiD mutant on nitrite-containing
medium was observed (Fig. 2). Moreover, a reduction in anaerobic
fermentative growth by the ywiD mutant was detected. From
these results, we conclude that ywiD is generally important
for anaerobic metabolism of B. subtilis. From these results
and others presented below, we have renamed ywiD
arfM (anaerobic respiration and fermentation modulator).
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 2.
Aerobic (A) and anaerobic fermentative (B) and nitrite
(C) and nitrate (D) respiratory growth of B. subtilis
wild-type JH642 ( ) and the arfM mutant MMB104 ( ).
The minimal medium is described in Materials and Methods. Growth was
monitored by determination of the OD578 at the indicated
time points. Values reported are the averages from at least five
independent experiments performed in triplicate.
|
|
Mutation of arfM leads to reduced nitrite reductase
activity.
To differentiate between the arfM influence
on various anaerobic respiratory systems, cell extracts prepared from
wild-type B. subtilis and the arfM mutant grown
under various anaerobic growth conditions were compared for respiratory
nitrate reductase and nitrite reductase activity. No significant
influence of arfM on respiratory nitrate reductase activity
was observed (Table 2). This is in
agreement with the anaerobic growth behavior of the arfM
mutant on nitrate-containing medium. Interestingly, reduced nitrate
reductase activity was observed in the wild-type and the arfM mutant under nitrite dissimilatory conditions,
indicating nitrite repression (data not shown). In contrast to the
results of the nitrate reductase activity measurements, the nitrite
reductase activity was reduced approximately fivefold in the
arfM mutant, indicating the participation of arfM
in nasDE expression or nitrite reductase formation or
activity (Table 2).
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Respiratory nitrate and nitrite reductase activities in
cell extracts prepared from wild-type B. subtilis and the
arfM mutanta
|
|
HPLC analysis of fermentation products of arfM
mutant.
The amounts of the fermentation products lactate, acetate,
and 2,3-butanediol excreted by wild-type B. subtilis and an
arfM mutant were compared using HPLC analysis
(3). The amount of accumulated lactate and 2,3-butanediol
formed under all anaerobic growth conditions tested using glucose and
pyruvate as carbon sources was found to be reduced by approximately
60% for the arfM mutant compared to the wild-type strain,
taking the difference in growth yield between the two strains into
account (data not shown). No obvious change in acetate production was
observed. These results indicate an impact of arfM on
lactate and 2,3-butanediol formation and provide an explanation for the
arfM growth phenotype under anaerobic fermentative conditions.
arfM gene is involved in anaerobic
nasDE but not narGHJI expression.
To
test the influence of arfM on the transcription of various
genes encoding anaerobic metabolism enzymes, the expression of reporter
gene fusions of corresponding promoter regions with lacZ
were tested. -Galactosidase activities in wild-type B. subtilis and the arfM mutant grown under various
anaerobic conditions were compared. In agreement with the growth
phenotypes and enzymatic activities, no significant influence of
arfM on narGHJI expression was found (Table
3). In agreement with the enzyme
activity measurements, less narGHJI expression was observed
in the presence of nitrite, again indicating nitrite inhibition (Table
3). The arfM mutation significantly reduced nasDE
expression under all anaerobic growth conditions tested (Table 3). This
is in agreement with the observed growth phenotype and reduced nitrite
reductase activities of an arfM mutant.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Influence of regulatory genes resDE, fnr, and
arfM on transcription of the nitrate and nitrite
dissimilatory loci narGHJI and nasDE and the
flavohemoglobin gene hmpa
|
|
arfM gene is involved in anaerobic
hmp induction.
The hmp gene, encoding a
flavohemoglobin-like protein, is another target of redox regulation in
B. subtilis. The importance of resDE and nitrite
for anaerobic induction of hmp was described previously
(8). As outlined by LaCelle et al., the observed anaerobic
induction by nitrate is of an indirect nature (8). Respiratory nitrate reductase reduces nitrate to nitrite. The nitrite
formed subsequently induces hmp transcription via a still unknown regulatory system. Anaerobic hmp transcription was
found to be significantly reduced in an arfM mutant under
all growth conditions tested (Table 3).
arfM gene is involved in anaerobic induction of the
fermentation loci lctEP and alsSD.
The involvement of fnr, resDE, alsR,
and the respiratory nitrate reductase operon narGHJI in
oxygen-, pH-, and nitrate-dependent lctEP and
alsSD expression control was described previously
(3). However, fnr was only partially
responsible for anaerobic lctEP and alsSD
expression. No obvious oxygen or nitrate regulation was observed for
pta (3).
Reporter gene fusions of the promoter regions of the fermentation loci
lctEP, alsSD, and pta were tested for
the participation of arfM in their anaerobic expression. A
significant reduction in anaerobic lctE-lacZ
expression in an arfM mutant compared to wild-type B. subtilis was observed (Table 4). The
previously observed nitrate repression of lctE transcription
remained mainly unchanged (3).
alsS-lacZ expression was also found to be reduced in the arfM mutant under conditions of fermentative as well
as nitrate and nitrite dissimilatory growth. Again, nitrate repression of alsS-lacZ expression remained mainly
unaffected (Table 4).
Consequently, arfM contributes to anaerobic lctEP
and alsSD induction. No arfM involvement in
pta transcription was observed (Table 4). This result was
expected because pta transcription lacks obvious redox
regulation (3). These results identify arfM as
an important component of the redox regulatory system in B. subtilis. To investigate the relationship of arfM to
the other known members of the redox system encoded by resDE
and fnr, the regulation of arfM transcription was investigated.
resDE-fnr-dependent anaerobic arfM
induction.
Redox-dependent arfM transcription was
investigated using arfM-lacZ fusions. As shown in
Table 5, arfM was exclusively
expressed anaerobically. Comparable values of
arfM-lacZ expression were obtained for
fermentative and nitrate respiratory growth, while the presence of
nitrite resulted in a 50% reduction in arfM expression. A
similar reduction in anaerobic gene expression by the presence of
nitrite was also observed for narGHJI transcription (see
above).
Anaerobic arfM expression was completely dependent on the
presence of intact fnr (Table 5). The involvement of
fnr in arfM transcription was expected because
the 5' region of arfM harbors a conserved potential Fnr
binding site (TGTGA-6N-TCACT). An arfM promoter analysis is
described below. Anaerobic expression of arfM in a
resDE mutant was found to be significantly reduced (Table 5). However, resDE-independent expression of fnr
from an IPTG-inducible Pspac promoter in a resDE
mutant restored anaerobic arfM expression to almost the
wild-type level (Table 5). These findings indicate that the observed
role of resDE in arfM expression is indirect, via
resDE-dependent fnr induction. As observed for
various other regulatory genes, arfM represses its own
expression (Table 5).
These results suggest that the anaerobic induction of various genes of
anaerobic metabolism is mediated by a regulatory cascade consisting of
an unknown signal, resDE, fnr, and
arfM. Investigation of the contribution of this potential
regulatory cascade to the induction of various anaerobic loci using
regulatory double mutants in combination with reporter gene fusions is
described below.
Analysis of fnr-dependent arfM
promoter.
Primer extension analysis revealed a single 5' end for
arfM mRNA (data not shown). A transcriptional start site
located 25 bp from the translational start was determined (Fig. 1).
Signal intensities during the primer extension experiments varied
depending on the growth conditions used for the B. subtilis
employed for RNA isolation. In agreement with the reporter gene
fusions, primer extension signals were only observed with RNA prepared
from anaerobically grown B. subtilis. Centered at 40.5 bp
upstream of the transcriptional start, we found a DNA sequence
(TGTGA-N6-TCACT) with a high degree of sequence
identity to potential B. subtilis Fnr binding sites.
However, no direct experimental proof was available for the function of
this promoter element in B. subtilis. Therefore, the upstream half of the palindromic sequence was mutated from
5'-TGTGA-3' to 5'-CCTGA-3'. As shown in Table 5,
the mutations in the putative Fnr-box (strain MMB25) completely
abolished anaerobic induction of arfM. These results
confirmed the complete fnr dependence of anaerobic
arfM induction and experimentally verified the
5'-TGTGA-N6-TCACT-3' sequence as the
Fnr-box.
Role of resDE-fnr-arfM regulatory cascade for
differential anaerobic gene expression.
The results from the
experiments described above and from a previous study of B. subtilis hemN and hemZ expression led us to propose a
model for arfM function (Fig.
3). The protein encoded by
arfM is currently the last part of a regulatory chain in
B. subtilis responsible for adaptation to anaerobic growth
conditions. The signal of low oxygen tension is measured by a still
unknown receptor and transferred directly or indirectly to the
two-component regulatory system ResDE. Subsequently ResDE directly
activates fnr transcription (14). Moreover,
several other genes of anaerobic metabolism such as hmp and
nasDE are influenced directly in their expression by
resDE.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 3.
Proposed regulatory cascade involved in the induction of
gene expression under low oxygen tension conditions in B.
subtilis (2, 3, 6, 8, 13, 13, 16, 20, 21). Various
stimuli (oxygen tension, nitrate, nitrite, and pH) are transferred and
integrated via various regulatory loci (resDE,
fnr, arfM, and alsR) to
differentially control the expression of various metabolic target genes
encoding respiratory nitrate reductase (narGHJI),
nitrite reductase (nasDE), flavohemoglobin-like protein
(hmp), enzymes of lactate (lctEP) and
acetoin (alsSD) fermentation and heme biosynthesis
(hemN and hemZ).
|
|
Fnr directly activates the most efficient anaerobic mode of ATP
generation, nitrate respiration, via induction of the nitrate reductase
operon narGHJI and nitrate/nitrite transporter genes. Fnr
also activates arfM transcription. Finally, arfM
modulates the expression of genes encoding proteins which further
sustain nitrate respiration, such as heme biosynthesis genes
(6). It also enhances alternative modes of ATP generation
to nitrate respiration such as fermentation and nitrite dissimilation.
This level of the molecular response to anaerobiosis is further fine
tuned by additional environmental and cellular stimuli mediated by
additional unknown redox regulatory components, by a pH-responding
system, and by nitrate as well as nitrite regulatory systems
(15).
In order to obtain further evidence for this regulatory model (Fig. 3),
double mutant strains defective in resDE, fnr, or arfM were constructed, and the expression patterns of
lacZ reporter gene fusions with all investigated genes were
measured (Tables 3 and 4). Combination of the arfM mutation
with the fnr or resDE mutation did not
significantly change their already detrimental effects on
narG-lacZ expression (Table 3). As expected,
combining the arfM and fnr mutations resulted in
similar -galactosidase activity values resulting from
nasD-lacZ and hmp-lacZ
expression compared to expression in a simple fnr mutant
(Table 3). Combination of the arfM and resDE
mutations led to a complete loss of transcription from the
nasD and hmp promoters. These additive effects
were expected due to the significant direct role of resDE in
nasDE and hmp transcription.
The comparable -galactosidase activity values derived from the
lctE-lacZ fusion in the arfM and
fnr single mutants as well as the arfM fnr double
mutant are in good agreement with our cascade regulatory model (Table
4). The lower values from the resDE mutants are in agreement
with the previously determined independent role of resDE in
lctE expression (3). This conclusion is further sustained by the significantly reduced lctE expression in a
resDE arfM double mutant. The similar values obtained for
alsS-lacZ expression in resDE,
fnr, and arfM mutants and the arfM
resDE double mutant are in agreement with the proposed regulatory
cascade. Surprisingly, no arfM fnr double mutant carrying an
alsS-lacZ fusion was obtained. The reason remains unclear.
Previously, we described the nitrate repression of
lctE-lacZ and alsS-lacZ
mediated by the presence of intact narGHJI. In agreement
with the lack of arfM influence on narGHJI
transcription, the arfM mutation did not significantly
affect nitrate repression. Combination of an arfM mutation
with either an fnr or a resDE mutation resulted
in the loss of nitrate repression due to decreased narGHJI
expression. For similar reasons, anaerobic
nasD-lacZ and hmp-lacZ
expression in the presence of nitrate was reduced in an arfM
mutant and totally abolished in an fnr mutant (Table 3). In
the fnr mutant, narGHJI expression is greatly
reduced. The synthesis of the strongly stimulatory nitrite from nitrate
by nitrate reductase is missing. Since the arfM mutation has
no effect on narGHJI expression, the combination of both
regulatory mutations resulted in the expected fnr
mutant-like expression pattern.
Similar to findings for B. subtilis hemN and hemZ
transcription, nasDE, hmp, lctEP, and
alsSD transcription is subject to resDE-fnr-arfM cascade regulation.
| |
ACKNOWLEDGMENTS |
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." 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 in Chicago) for the gift of B.
subtilis mutants. We thank R. K. Thauer
(Max-Planck-Institut, Marburg, Germany) for continuous support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie, Technische Universität Braunschweig,
Spielmannstr.7, 38106 Braunschweig, Germany. Phone: 49(0)531-3915801.
Fax: 49(0)531-3915854. E-mail: d.jahn{at}tu-bs.de.
Present address: Department of Molecular Biology and Biotechnology,
The University of Sheffield, Firth Court, Western Bank, Sheffield S10
2TN, United Kingdom.
| |
REFERENCES |
| 1.
|
Chung, C. T., and R. H. Miller.
1988.
A rapid and convenient method for the preparation and storage of competent bacterial cells.
Nucleic Acids Res.
16:3580[Free Full Text].
|
| 2.
|
Cruz Ramos, H.,
L. Boursier,
I. Moszer,
F. Kunst,
A. Danchin, and P. Glaser.
1995.
Anaerobic transcription activation in Bacillus subtilis: identification of distinct FNR-dependent and -independent regulatory mechanisms.
EMBO J.
14:5984-5994[Medline].
|
| 3.
|
Cruz Ramos, H.,
T. Hoffmann,
M. Marino,
E. Presecan-Siedel,
H. Nedjari,
O. Dreesen,
R. Longin,
P. Glaser, and D. Jahn.
2000.
The fermentative metabolism of Bacillus subtilis: physiology and regulation of gene expression.
J. Bacteriol.
182:3072-3080[Abstract/Free Full Text].
|
| 4.
|
Hoffmann, T.,
N. Frankenberg,
M. Marino, and D. Jahn.
1998.
Ammonification in Bacillus subtilis utilizing dissimilatory nitrite reductase is dependent on resDE.
J. Bacteriol.
180:186-189[Abstract/Free Full Text].
|
| 5.
|
Hoffmann, T.,
B. Troup,
A. Szabo,
C. Hungerer, and D. Jahn.
1995.
The anaerobic life of Bacillus subtilis: cloning and characterization of the genes encoding the respiratory nitrate reductase system.
FEMS Microbiol. Lett.
131:219-225[CrossRef][Medline].
|
| 6.
|
Homuth, G.,
A. Rompf,
W. Schumann, and D. Jahn.
1999.
Characterization of Bacillus subtilis hemZ.
J. Bacteriol.
181:5922-5929[Abstract/Free Full Text].
|
| 7.
|
Kunst, F., and G. Rapoport.
1995.
Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis.
J. Bacteriol.
177:2403-2407[Abstract/Free Full Text].
|
| 8.
|
LaCelle, M.,
M. Kumano,
K. Kurita,
K. Yamane,
P. Zuber, and M. M. Nakano.
1996.
Oxygen-controlled regulation of the flavohemoglobin gene in Bacillus subtilis.
J. Bacteriol.
178:3803-3808[Abstract/Free Full Text].
|
| 9.
|
Mach, H.,
M. Hecker, and F. Mach.
1988.
Physiological studies on cAMP synthesis in Bacillus subtilis.
FEMS Microbiol. Lett.
52:189-192[CrossRef].
|
| 10.
|
Miller, J. H.
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 11.
|
Murphy, E.
1985.
Nucleotide sequence of a spectinomycin adenyltransferase AAD(9) determinant from Staphylococcus aureus and its relationship to AAD(3")(9).
Mol. Gen. Genet.
200:33-39[CrossRef][Medline].
|
| 12.
|
Nakano, M. M.,
Y. P. Dailly,
P. Zuber, and D. P. Clark.
1997.
Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth.
J. Bacteriol.
179:6749-6755[Abstract/Free Full Text].
|
| 13.
|
Nakano, M. M.,
T. Hoffmann,
Y. Zhu, and D. Jahn.
1998.
Nitrogen and oxygen regulation of Bacillus subtilis nasDEF encoding NADH-dependent nitrite reductase by TnrA and ResDE.
J. Bacteriol.
180:5344-5350[Abstract/Free Full Text].
|
| 14.
|
Nakano, M. M.,
Y. Zhu,
M. LaCelle,
X. Zhang, and F. M. Hulett.
2000.
Interaction of ResD with regulatory regions of anaerobically induced genes in Bacillus subtilis.
Mol. Microbiol.
37:1198-1207[CrossRef][Medline].
|
| 15.
|
Nakano, M. M., and P. Zuber.
1998.
Anaerobic growth of a "strict aerobe" (Bacillus subtilis).
Annu. Rev. Microbiol.
52:165-190[CrossRef][Medline].
|
| 16.
|
Nakano, M. M.,
P. Zuber,
P. Glaser,
A. Danchin, and F. M. Hulett.
1996.
Two-component regulatory proteins ResD-ResE are required for transcriptional activation of fnr upon oxygen limitation in Bacillus subtilis.
J. Bacteriol.
178:3796-3802[Abstract/Free Full Text].
|
| 17.
|
Perego, M.
1993.
Integrational vectors for genetic manipulation in Bacillus subtilis, p. 615-624.
In
A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. ASM Press, Washington, D.C.
|
| 18.
|
Presecan-Siedel, E.,
A. Galinier,
R. Longin,
J. Deutscher,
A. Danchin,
P. Glaser, and I. Martin-Verstraete.
1999.
Catabolite regulation of the pta gene as part of the carbon flow pathways in Bacillus subtilis.
J. Bacteriol.
181:6889-6897[Abstract/Free Full Text].
|
| 19.
|
Presecan, E.,
I. Moszer,
L. Boursier,
H. C. Cruz Ramos,
V. de la Fuente,
M. F. Hullo,
C. Lelong,
S. Schleich,
A. Sedowska,
B. H. Song,
G. Villani,
F. Kunst,
A. Danchin, and P. Glaser.
1997.
The Bacillus subtilis genome from gerBC (311 degrees) to licR (334 degrees).
Microbiology
143:3318-3328.
|
| 20.
|
Renna, M. C.,
N. Najimudin,
L. R. Winik, and S. A. Zahler.
1993.
Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin.
J. Bacteriol.
175:3863-3875[Abstract/Free Full Text].
|
| 21.
|
Sun, G.,
E. Sharkova,
R. Chesnut,
S. Birkey,
M. F. Duggan,
A. Sorokin,
P. Pujic,
S. D. Ehrlich, and F. M. Hulett.
1996.
Regulators of aerobic and anaerobic respiration in Bacillus subtilis.
J. Bacteriol.
178:1374-1385[Abstract/Free Full Text].
|
Journal of Bacteriology, December 2001, p. 6815-6821, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6815-6821.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
