Download - Wiley Online Library

FEMS Microbiology Letters 157 (1997) 1^7
MiniReview
Adaptation of Bacillus subtilis to oxygen limitation
Michiko M. Nakano *, F. Marion Hulett
a;
a
b
b
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, LA 71130-3932, USA
Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA
Received 18 August 1997; revised 15 September 1997; accepted 18 September 1997
Abstract
Bacillus subtilis grows anaerobically by at least two different pathways, respiration using nitrate as an electron acceptor and
fermentation in the absence of electron acceptors. Regulatory mechanisms have evolved allowing cells to shift to these
metabolic capabilities in response to changes in oxygen availability. These include transcriptional activation of fnr upon oxygen
limitation, a process requiring the ResD-ResE two-component signal transduction system that also regulates aerobic
respiration. FNR then activates transcription of other anaerobically induced genes including the narGHJI operon which
encodes a respiratory nitrate reductase. Genes involved in fermentative growth are controlled by an unidentified FNRindependent regulatory pathway.
Keywords: Bacillus subtilis ;
Nitrate respiration; Fermentation; Signal transduction; Anaerobic gene regulation
1. Introduction
2. Mode of anaerobic growth in B. subtilis
The Gram-positive, spore-forming bacterium Bais one of the most studied and best
characterized organisms used in basic and applied
microbiological research. B. subtilis has been generally considered a strict aerobe for many years. Recent studies that have established the conditions
under which B. subtilis grows anaerobically have
been followed by the identi¢cation of several genes
that function or are expressed under conditions of
anaerobiosis. These recent ¢ndings have uncovered
regulatory pathways governing anaerobic gene regulation. This review will summarize the accumulation
of knowledge concerning anaerobiosis in B. subtilis.
At present, two modes of anaerobic growth have
been described in B. subtilis. One is anaerobic respiration using nitrate as an electron acceptor [1^9]. B.
subtilis has apparently very weak, if any, fumaratedependent respiration [8,10]. A recent study showed
that the wild-type and the narGHJI mutant grow
anaerobically on nitrite and generate ammonia, suggesting that B. subtilis grows by nitrite respiration
(T. Ho¡man and D. Jahn, personal communication).
There have been no reports of B. subtilis utilizing
other, alternative electron acceptors such as DMSO
and TMAO which are known to be used as terminal
electron acceptors in Escherichia coli. The other
mode of anaerobic growth in B. subtilis is fermentation [10].
cillus subtilis
* Corresponding author. Tel.: +1 (318) 675 5158; Fax:
+1 (318) 675 5180; E-mail: [email protected]
0378-1097/97/$32.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 3 6 - 9
2
M.M. Nakano, F.M. Hulett / FEMS Microbiology Letters 157 (1997) 1^7
2.1. Nitrate respiration
Although taxonomists have used nitrate reduction
to nitrite as a diagnostic character for B. subtilis for
many years [11], the ¢rst physiological study suggesting that B. subtilis could grow anaerobically showed
that membrane-bound nitrate reductase was present
only under low culture aeration [12]. We know now
that B. subtilis has two distinct nitrate reductases,
one is assimilatory and the other respiratory
[1,3,13]. In retrospect, the nitrate reductase reported
above should be the respiratory one encoded by
narGHJI since the enzyme was shown later to be
induced under anaerobiosis and not under the control of the global nitrogen regulatory network that
operates in B. subtilis and which governs the expression of genes encoding the assimilatory nitrate reductase (see below).
More intensive and systematic studies have been
undertaken over the past 3 years to examine the
ability of B. subtilis cells to grow anaerobically.
Most, if not all, studies were inspired by a passage
in a review written by Priest [6]: ``Although B. subtilis is generally regarded as an aerobe, it can grow
and sporulate slowly under strict anaerobic conditions. Given glucose, with nitrate as a terminal electron acceptor, it grows strongly anaerobically''. The
anaerobic growth of B. subtilis on nitrate is truly
respiratory since B. subtilis was able to grow anaerobically in the presence of nitrate with glycerol as a
sole carbon source [10]. The ¢rst mutation which
resulted in defective nitrate respiration was shown
to be the homologue of moaA which is required for
biosynthesis of the molybdenum cofactor for nitrate
reductase [2]. The study also provided evidence for at
least two nitrate reductases in B. subtilis, one of
which is involved in nitrate respiration. The work
was followed by the identi¢cation of genes, primarily
by members of the Bacillus genome sequencing project consortium, whose products function in nitrate
respiration. These include narGHJI, narK, fnr, resD
and resE which will be described later.
2.2. Fermentation
Some bacteria such as E. coli undergo fermentation if no electron acceptor is available. In fermentation, NADH generated by glycolysis cannot be re-
oxidized by electron transport reactions. Instead
NAD‡ is generated using endogenous electron acceptors produced during metabolism of pyruvate
while ATP is generated by substrate-level phosphorylation unlike the case of respiration where proton
potential is used to synthesize ATP. Fermentation,
as carried out by E. coli, has been described from the
viewpoint of redox balance in a review by Clark [14].
The fermentation pathways in B. subtilis are not well
understood. Glucose fermentation in B. subtilis is
poor if it occurs at all [8^10]. However, B. subtilis
cells grow by fermentation in the presence of glucose
and pyruvate, or alternatively with glucose and a
mixture of 20 amino acids [10]. The reasons why B.
subtilis cannot ferment either glucose or pyruvate
e¤ciently (unlike E. coli) and why pyruvate enhances
glucose fermentation are unknown. NMR analysis
showed that fermentation products in B. subtilis include acetate, acetoin, ethanol, lactate, succinate and
2,3-butanediol, indicating a mixed acid fermentation
[10].
3. Genes involved in anaerobiosis
3.1. Nitrate respiration
3.1.1. narGHJI and moaA
As mentioned above, the narGHJI genes encoding
subunits of a respiratory nitrate reductase and moaA
involved in biosynthesis of the reductase-associated
molybdenum cofactor have been isolated from B.
subtilis. Of two classic mutations (narA and narB)
which cause defects in nitrate assimilation, one also
a¡ects nitrate respiration. One of the mutations, formerly narB, was assigned to the nasBCDEF operon,
encoding subunits of nitrate/nitrite reductases and an
enzyme involved in the synthesis of siroheme, a cofactor of nitrite reductase [13]. The other locus, formerly narA, was shown to include narQ and moaA
[2]. The putative narQ product showed similarity to
FdhD which is required for formate dehydrogenase
activity in E. coli. MoaA showed homology to E. coli
MoaA, a protein required for biosynthesis of the
molybdenum cofactor for nitrate reductase and formate dehydrogenase [15]. Mutations in either nasBCDEF or moaA resulted in the cell's inability to use
nitrate as a sole nitrogen source, indicating that each
M.M. Nakano, F.M. Hulett / FEMS Microbiology Letters 157 (1997) 1^7
has a role in nitrate assimilation. A moaA mutant,
unlike nasBCDEF mutants, was unable to grow
anaerobically in the presence of nitrate. In agreement
with this, a nasB mutant retained respiratory nitrate
reductase activity, in contrast to a mutation in moaA
that resulted in loss of both activities [2]. This indicated that there are two distinct nitrate reductases in
B. subtilis, one encoded by the nasBC genes which is
involved in nitrate assimilation and the other required for nitrate respiration, the gene yet to be identi¢ed. MoaA is essential for the activity of both the
assimilatory and the respiratory enzymes.
The narGHJI genes encoding the putative respiratory nitrate reductase were isolated as part of the
Bacillus genome sequencing project [1] and by an
independent search using oligonucleotides deduced
from a conserved amino acid sequence of a respiratory nitrate reductases from E. coli [3]. As expected,
mutations in the narGHJI genes abolished nitrate
respiration [3,4]. By analogy with the homologous
system of E. coli, narG, H and I [16] probably encode
three subunits of the enzyme and the narJ product
likely functions in the assembly of the enzyme complex. These four genes constitute the narGHJI operon which also contains a putative FNR-binding site
in its regulatory region.
3.1.2. narK
The narK
gene resides upstream of the narGHJI
operon [1]. NarK of E. coli was shown to function in
nitrite extrusion during nitrate respiration [17,18].
narK is the ¢rst gene of a dicistronic operon that
also contains the fnr gene. The B. subtilis narK
gene was shown to be able to complement the defect
of an E. coli narK mutant [1].
3.1.3. fnr
fnr, the second gene of the narK-fnr operon, encodes a protein homologous to E. coli FNR, the
global anaerobic gene regulator. Disruption of fnr
abolished nitrate reductase activity under anaerobic
conditions and nitrate respiration [1]. A cysteine residue cluster present near the N-terminus of E. coli
FNR, which is thought to have a role in modulating
the activity of the protein by a mechanism involving
iron [19], was also found in the C-terminal end of B.
subtilis FNR. B. subtilis FNR, like E. coli FNR, is a
member of the E. coli CAP activator family. Putative
3
FNR binding sites (TGTGAN6 TCACA) in B. subtilis were identi¢ed by aligning the sequences of pro-
moter regions associated with FNR-controlled genes.
The sequence is identical to the consensus sequence
for the target site for E. coli CAP but not to the E.
coli FNR binding site (TTGATN4 ATCAA). In fact,
the FNR site of B. subtilis was shown to be recognized by E. coli CAP since the transcription from the
narK promoter was observed to be activated 50-fold
in E. coli by the cAMP-CAP complex [1]. Unlike E.
coli, where fnr expression is weakly repressed under
anaerobic conditions by FNR itself, expression of B.
subtilis fnr is strongly activated by oxygen limitation.
3.1.4. resD and resE
The resD and resE genes were also identi¢ed by
the Bacillus subtilis sequencing project [20]. Sequence
analysis suggested that ResE, a histidine sensor kinase, and ResD, a response regulator, are members of
the two-component signal transduction family of
proteins. resD and resE constitute the resA operon
together with three upstream genes (resA, B and C)
which were shown to be essential genes encoding
proteins with similarity to those that function in cytochrome c biogenesis. resD mutant phenotypes include streptomycin resistance, lack of production of
aa3 or caa3 terminal oxidases, acid accumulation
when grown with glucose as a carbon source, and
a requirement for 6-carbon compounds as sources
of energy. All of these properties implicate ResDE
as having a role in respiration [7,21]. Interestingly,
the ResD-ResE signal transduction system is not
only essential for aerobic respiration but also for
anaerobic respiration using nitrate as a terminal electron acceptor [7]. The requirement for ResD-ResE
both for aerobic and anaerobic respiration in B. subtilis is novel since most genes involved in aerobic
respiration in E. coli are negatively regulated by either FNR, ArcA-ArcB two-component regulatory
system or by both while, in contrast, genes required
for anaerobic respiration are activated by these regulatory proteins [22]. The regulatory role of ResDE
in aerobic and anaerobic respiration will be discussed
later in detail.
3.1.5. ftsH
ftsH is known
as a member of the AAA family
(ATPases associated with a variety of cellular activ-
M.M. Nakano, F.M. Hulett / FEMS Microbiology Letters 157 (1997) 1^7
4
E. coli FtsH was suggested to funcftsH
gene of B. subtilis, identi¢ed by the Bacillus genome
ities) of proteins.
4. Regulatory pathways for nitrate respiration
tion as a chaperone or zinc protease [23]. The
Two
resDE
loci,
fnr,
and
which
encode
factors
sequencing project [24], was shown to be the site of
that control the expression of genes required for ni-
mutations
trate respiration have been identi¢ed. A regulatory
conferring
extremely
pleiotropic
pheno-
resDE
types which include high sensitivity to salt and heat
pathway for nitrate respiration governed by
stress, a defect in entry into the sporulation process,
and
and impaired secretion of exoproteins [25^27].
signal transduction system is required for anaerobic
ftsH
B.
mutants were also isolated among a collection of
subtilis mutants unable to grow by anaerobic fermentation [10]. ftsH was also shown to be required, to a
fnr
is summarized in Fig. 1. The ResD-ResE
induction of
fnr
transcription upon oxygen limita-
tion. FNR positively regulates other anaerobically-
narGHJI and narK. This sec-
induced genes such as
lesser extent, for nitrate respiration. The role of FtsH
tion describes how this conclusion was drawn from
in anaerobiosis and in other cellular processes re-
the experimental data.
mains to be elucidated.
3.1.6. ace
An ace mutant which lacks pyruvate dehydrogen-
4.1. The ResD-ResE signal transduction system is
required for transcription of fnr from an
fnr-speci¢c promoter
ase (PDH) activity is unable to grow by nitrate respiration, suggesting that PDH is involved in pyruvate catabolism in nitrate-respiring cells [10]. In
coli,
E.
either PFL or PDH can be used to catabolize
pyruvate in nitrate respiration [28].
ResD, and to a lesser extent ResE, are required for
aerobic respiration since they are essential for the
expression
of
genes,
such
resA, ctaA (required
petCBD operon (encod-
as
for HemA synthesis) and the
3.2. Fermentation
Few
B. subtilis
genes
required
for
fermentation
have been identi¢ed. Among the genes required for
nitrate respiration,
narGHJI
and
be dispensable for fermentation.
fnr were shown to
resD mutations af-
fect fermentation moderately, indicating that ResD
may
function
in
an
FNR-independent
regulatory
pathway controlling fermentation [10]. A search for
mutants that were unable to carry out fermentation
uncovered the
required
for
ftsH gene which was shown also to be
nitrate
respiration
(described
above)
[10]. A key issue for fermentation is how pyruvate
is metabolized to ful¢l the cells' need to reoxidize the
NADH produced by glycolysis. During fermentation
in most anaerobic bacteria, pyruvate is metabolized
by pyruvate formate lyase (PFL) instead of
which
generates
extra
NADH
[14,29].
PDH
However,
Fig. 1. Regulatory pathway of nitrate respiration in
Arrows
with
lines
indicate
directions
of
B. subtilis.
transcription.
Also
PDH is likely to be utilized most often, if not exclu-
shown are possible £ows of information to induce nitrate respira-
sively, for the conversion of pyruvate to acetyl CoA
tion. ResD phosphorylated by a ResE kinase activates the tran-
in
scription
B. subtilis fermentation since the ace mutant can-
not grow fermentatively [10].
of
the
resA
operon.
for the anaerobic induction of
ResD-phosphate
fnr
is
also
required
transcription from the
fnr-spe-
ci¢c promoter. FNR activates transcription of genes involved in
nitrate
respiration
such
as
the
narK
and
narG
operons.
produced from nitrate by nitrate reductase induces
tion.
Nitrite
hmp transcrip-
M.M. Nakano, F.M. Hulett / FEMS Microbiology Letters 157 (1997) 1^7
ing
subunits
encode
of
the
products
cytochrome
that
function
bf
complex),
in
that
respiration
Another
important
5
question
is
the
following.
If
[7].
ResD is a transcriptional activator, does it bind to
Since ResD/ResE are required for optimal aerobic
the regulatory regions of all ResD/E-controlled genes
and anaerobic respiration and transcription of
fnr
resABCDE, ctaA, petCBD and fnr ? If so,
fnr transcription regulated speci¢cally by oxy-
such as
is highly induced by anaerobiosis, one could imagine
how is
that the ResD/E system occupies a higher epistatic
gen limitation ? If ResD is not a transcriptional acti-
level than FNR in the anaerobic regulon. Expression
vator for
of
fnr was
fnr,
what is the activator, expression of
shown to be regulated by two mechanisms
which may require ResD and ResE. There are no
that exert their e¡ects at two promoters, the operon
obvious structural similarities observed among the
narK
promoter located upstream of
and the
fnr-spe-
regulatory
regions
of
the
ResDE-controlled
genes,
ci¢c, intergenic promoter. The transcription at the
although the absence of such a sequence does not
narK
operon promoter is dependent on FNR and
always exclude the possibility that ResD binds to
the intergenic promoter was activated by anaerobio-
all of them. In an attempt to gain a further under-
sis independently of FNR [1]. This FNR-independent activation of
fnr
by anaerobiosis was shown to
be dependent on the ResD-ResE signal transduction
lacZ fused to
the fnr-speci¢c promoter in the wild-type, resD, resE,
and resDE mutants [5]. Mutations in resD or resE
completely abolished anaerobic induction of fnr, insystem by examining the expression of
standing of
tory
site
fnr
regulation by ResD/E, a
controlled
by
ResDE
under
cis-regula-
anaerobiosis
was identi¢ed. Deletion and mutational analysis of
the
fnr
try
(TNACAAN2 TTGTNA)
promoter identi¢ed a region of dyad symmecentered
at
3
52.5
as
being required for anaerobic induction by ResD/E
(M. Nakano, unpublished result).
dicating ResD phosphorylated by a ResE kinase is
fnr
required for transcriptional activation of
upon
oxygen limitation. FNR thus produced activates expression of the
moter.
An
anaerobiosis,
tion of
fnr,
narK-fnr
additional
in
operon from the
role
addition
of
to
ResD
narK
and
pro-
ResE
transcriptional
4.2. FNR is a regulator required for other
anaerobically induced genes
in
activa-
Unlike its
E. coli
B. subtilis fnr
homologue,
is in-
duced by anaerobiosis as described above. Another
level of control that modulates FNR activity could
was suggested [5].
The activation of the ResD-ResE signal transduc-
be exerted in response to changes in oxygen levels by
tion system could be regulated at two levels : expres-
virtue of the iron-cysteine clusters and their redox
sion of the
resDE
genes and generation of a signal to
state as has been proposed for
E. coli
FNR. Putative
which ResE responds leading to autophosphoryla-
FNR binding sites (TGTGAN6 TCACA) were found
tion.
in promoter regions of ¢ve genes/operons in
resD
resA
Studying
the
resE
and
two
are
in
isolation
mainly
is
di¤cult,
transcribed
from
as
the
operon promoter which is, itself, dependent of
tilis,
position
3
41.5 [1]. These genes are
were also shown to
ywcJ, ywiC
be transcribed from an intergenic promoter [7]. The
between the
ResD-phosphate.
resD
activity of the
the
culture
possibility
from the
resD
not
resD
resE
promoter is weak at least under
conditions
is
and
so
far
excluded
tested,
that
the
although
the
transcription
promoter is induced more strongly
under as yet unidenti¢ed conditions. A recent study
shows that
resE
resA
transcription, and, therefore,
B. sub-
and in each case, the sequence is centered at
ywiD, the
narK and narG
and
narGHJI, narK,
last two genes, located
operons and divergently
transcribed, likely share a putative FNR binding site
but their function in anaerobiosis is unknown. The
ywcJ
gene product is similar to the
E. coli nirC
gene
product, the function of which is not known. Among
these genes,
narGHJI
and
narK
were shown to re-
resD
quire FNR for their anaerobic induction [1]. It is
expression, is higher in cells grown anae-
likely that FNR regulates transcription by interact-
robically than those grown aerobically (M. Nakano,
ing with the FNR-binding site located in their pro-
unpublished results). One important problem to be
moter regions, although there is no report yet show-
investigated is the nature of the signal that activates
ing
the ResDE system.
sequences.
and
the
binding
of
FNR
to
these
cis-regulatory
6
M.M. Nakano, F.M. Hulett / FEMS Microbiology Letters 157 (1997) 1^7
4.3. Nitrite-induced anaerobic expression of the hmp
gene
Another mode of anaerobic gene regulation was
found in B. subtilis in a search for anaerobically
induced genes. [4]. One such gene, encoding Hmp
protein, is a hemoglobin-like protein that belongs
to a family of two-domain £avohemoproteins [30]
which have been identi¢ed in various organisms
such as E. coli and Saccharomyces cerevisiae.
Although Hmp is dispensable for anaerobic growth,
the expression of hmp is strongly induced by anaerobiosis and the induction is dependent on NarGHJI,
FNR and ResD-ResE [4]. The requirement of FNR
and NarGHJI for the expression was completely bypassed by the addition of nitrite in the culture medium, indicating that fnr is required for expression of
narGHJI, which converts nitrate to nitrite, leading to
induction of hmp expression. In contrast, the requirement for ResDE was not bypassed by nitrite. The
additional role of ResDE for hmp expression remains
to be uncovered. In E. coli, dual sensors NarQ and
NarX and the dual regulators NarL and NarP are
involved in nitrate/nitrite regulation [31]. How nitrite
regulates the expression of B. subtilis hmp is unknown.
5. Conclusion
The natural habitat of B. subtilis is soil, an ecological realm that contains an abundance of anaerobic environments. Water-saturated soil crumbs larger
than 3 mm in radius were calculated to have no oxygen in their centers [32] and strictly anaerobic bacteria such as clostridia commonly exist in the upper
layer of soil [32]. Studies on anaerobiosis in B. subtilis have just begun, but studies presented in this
review have shown that the anaerobic life of B. subtilis has several unique features when compared to
the well-studied enteric systems. Most notable are
the anaerobic induction of the fnr expression and
the involvement of the ResDE signal transduction
system in both aerobic and anaerobic gene regulation. In E. coli, FNR and ArcA/B function as both
positive and negative regulators. No information is
available at present concerning whether some genes
in B. subtilis are negatively regulated by FNR and/or
ResD/E. How are genes involved in aerobic respiration repressed under anaerobiosis? The question is
intriguing since ResDE is required for aerobic and
anaerobic respiration and some regulatory mechanism to turn o¡ aerobic respiration should exist.
The investigation of gene expression in response to
oxygen limitation will likely uncover regulatory connections to other stress responses such as nutritional
stress and high cell density in B. subtilis, as has been
shown in the case of genetic competence, sporulation, motility, etc. Since B. subtilis has been widely
used as a host in the fermentation industry and oxygen de¢ciency in large-scale growth vessels is critical
for improved product yield, the work on anaerobiosis in B. subtilis will also provide an opportunity to
improve the organism's use in the biotechnology industry.
Acknowledgments
We thank Peter Zuber for valuable comments on
the manuscript. We apologize that many notable
studies could not be cited in this review due to the
citation limitations. The research at Louisiana State
University and University of Illinois at Chicago is
supported by NIH Grants GM45898 and
GM33471, respectively.
References
[1] Cruz Ramos, H., Boursier, L., Moszer, I., Kunst, F., Danchin, A. and Glaser, P. (1995) Anaerobic transcription activation in Bacillus subtilis : Identi¢cation of distinct FNR-dependent and -independent regulatory mechanisms. EMBO J.
14, 5984^5994.
[2] Glaser, P., Danchin, A., Kunst, F., Zuber, P. and Nakano,
M.M. (1995) Identi¢cation and isolation of a gene required
for nitrate assimilation and anaerobic growth of Bacillus subtilis. J. Bacteriol. 177, 1112^1115.
[3] Ho¡mann, T., Troup, B., Szabo, A., Hungerer, C. and Jahn,
D. (1995) The anaerobic life of Bacillus subtilis : Cloning of
the genes encoding the respiratory nitrate reductase system.
FEMS Microbiol. Lett. 131, 219^225.
[4] LaCelle, M., Kumano, M., Kurita, K., Yamane, K., Zuber, P.
and Nakano, M.M. (1996) Oxygen-controlled regulation of
£avohemoglobin gene in Bacillus subtilis. J. Bacteriol. 178,
3803^3808.
[5] Nakano, M.M., Zuber, P., Glaser, P., Danchin, A. and Hulett, F.M. (1996) Two-component regulatory proteins ResD-
M.M. Nakano, F.M. Hulett / FEMS Microbiology Letters 157 (1997) 1^7
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
ResE are required for transcriptional activation of fnr upon
oxygen limitation in Bacillus subtilis. J. Bacteriol. 178, 3796^
3802.
Priest, F.G. (1993) Systematics and ecology of Bacillus. In:
Bacillus subtilis and Other Gram-Positive Bacteria. Biochemistry, Physiology, and Molecular Genetics (Sonenshein, A.L.,
Hoch, J.A. and Losick, R., Eds.), pp. 3^16, American Society
for Microbiology, Washington, DC.
Sun, G., Sharkova, E., Chesnut, R., Birkey, S., Duggan,
M.F., Sorokin, A., Pujic, P., Ehrlich, S.D. and Hulett, F.M.
(1996) Regulators of aerobic and anaerobic respiration in Bacillus subtilis. J. Bacteriol. 178, 1374^1385.
Schirawski, J. and Unden, G. (1995) Anaerobic respiration of
Bacillus macerans with fumarate, TMAO, nitrate and nitrite
and regulation of the pathways by oxygen and nitrate. Arch.
Microbiol. 163, 148^154.
Shariati, P., Mitchell, W.J., Boyd, A. and Priest, F.G. (1995)
Anaerobic metabolism in Bacillus licheniformis NCIB 6346.
Microbiology 141, 1117^1124.
Nakano, M.M., Dailly, Y.P., Zuber, P. and Clark, D.P. (1997)
Characterization of anaerobic fermentative growth in Bacillus
subtilis : Identi¢cation of fermentation end products and genes
required for the growth. J. Bacteriol. (in press).
Gordon, R.E., Haynes, W.C. and Pang, C.H. (1973) The Genus Bacillus. Agricultural Handbook No. 427. US Department of Agriculture, Washington, DC.
Bohin, J.-P., Bohin, A. and Shae¡er, P. (1976) Increased nitrate reductase A activity as a sign of membrane alteration in
early blocked asporogenous mutants of Bacillus subtilis. Biochimie 58, 99^108.
Ogawa, K., Akagawa, E., Yamane, K., Sun, Z.-W., LaCelle,
M., Zuber, P. and Nakano, M.M. (1995) The nasB operon
and nasA gene are required for nitrate/nitrite assimilation in
Bacillus subtilis. J. Bacteriol. 177, 1409^1413.
Clark, D.P. (1989) The fermentation pathways of Escherichia
coli. FEMS Microbiol. Rev. 63, 223^234.
Rajagopalan, K.V. and Johnson, J.L. (1992) The pterin molybdenum cofactors. J. Biol. Chem. 267, 10199^10202.
Blasco, F., Iobbi, C., Giordano, G., Chippaux, M. and Bonnefoy, V. (1989) Nitrate reductase of Escherichia coli : completion of the nucleotide sequence of the nar operon and reassessment of the role of the alpha and beta subunits in iron
binding and electron transfer. Mol. Gen. Genet. 218, 249^256.
DeMoss, J.A. and Hsu, P.-Y. (1991) NarK enhances nitrate
uptake and nitrite excretion in Escherichia coli. J. Bacteriol.
173, 3303^3310.
Rowe, J.J., Ubbink-Kok, T., Molenaar, D., Konings, W.N.
and Driessen, A.J.M. (1994) NarK is a nitrite-extrusion system involved in anaerobic nitrate respiration by Escherichia
coli. Mol. Microbiol. 12, 579^586.
7
[19] Green, J., Sharrocks, A.D., Green, B., Geisow, M. and Guest,
J.R. (1993) Properties of FNR proteins substituted at each of
the ¢ve cysteine residues. Mol. Microbiol. 8, 61^68.
[20] Sorokin, A., Zumstein, E., Azevedo, V., Ehrlich, S.D. and
Serror, P. (1993) The organization of the Bacillus subtilis
168 chromosome region between the spoVA and serA genetic
loci, based on sequence data. Mol. Microbiol. 10, 385^395.
[21] Hulett, F.M. (1995) Complex phosphate regulation by sequential switches in Bacillus subtilis. In: Two-Component Signal
Transduction (Hoch, J.A. and Silhavy, T.J., Eds.), pp. 289^
302. Americal Society for Microbiology, Washington, DC.
[22] Gunsalus, R.P. and Park, S.-J. (1994) Aerobic-anaerobic gene
regulation in Escherichia coli : control by the ArcAB and Fnr
regulons. Res. Microbiol. 145, 437^450.
[23] Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka,
K., Niki, H., Hiraga, S. and Ogura, T. (1993) The Escherichia
coli FtsH protein is a prokaryotic member of a protein family
of putative ATPases involved in membrane functions, cell
cycle control, and gene expression. J. Bacteriol. 175, 1344^
1351.
[24] Ogasawara, N., Nakai, S. and Yoshikawa, H. (1994) Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin. DNA Res.
1, 1^14.
[25] Deuerling, E., Paeslack, B. and Shumann, W. (1995) The ftsH
gene of Bacillus subtilis is transiently induced after osmotic
and temperature shift. J. Bacteriol. 177, 4105^4112.
[26] Deuerling, E., Mogk, A., Richter, C., Purucker, M. and Schumann, W. (1997) The ftsH gene of Bacillus subtilis is involved
in major cellular processes such as sporulation, stress adaptation and secretion. Mol. Microbiol. 23, 921^933.
[27] Lysenko, E., Ogura, T. and Cutting, S.M. (1997) Characterization of the ftsH gene of Bacillus subtilis. Microbiology 143,
971^978.
[28] Kaiser, M. and Sawers, G. (1994) Pyruvate formate-lyase is
not essential for nitrate respiration by Escherichia coli. FEMS
Microbiol. Lett. 117, 163^168.
[29] Lin, E.C.C. and Iuchi, S. (1991) Regulation of gene expression
in fermentative and respiratory systems in Escherichia coli and
related bacteria. Annu. Rev. Genet. 25, 361^387.
[30] Andrews, S.C., Shipley, D., Keen, J.N., Findlay, J.B.C., Harrison, P.M. and Guest, J.R. (1992) The haemoglobin-like protein (HMP) of Escherichia coli has ferrisiderophore reductase
activity and its C-terminal domain shares homology with ferredoxin NADP‡ reductases. FEBS Lett. 302, 247^252.
[31] Stewart, V. (1993) Nitrate regulation of anaerobic respiratory
gene expression in Escherichia coli. Mol. Microbiol. 9, 425^
434.
[32] Paul, E.A. and Clark, F.E. (1989) Soil Microbiology and Biochemistry. Academic Press, San Diego, CA.