JOURNAL OF BACTERIOLOGY, Mar. 2001, p. 1938–1944 Vol. 183, No.

6
0021-9193/01/$04.00⫹0 DOI: 10.1128/JB.183.6.1938–1944.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Involvement of ResE Phosphatase Activity in Down-Regulation

of ResD-Controlled Genes in Bacillus subtilis during
Aerobic Growth
MICHIKO M. NAKANO* AND YI ZHU
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology,
Beaverton, Oregon 97006
Received 15 September 2000/Accepted 20 December 2000

The ResD-ResE signal transduction system is required for aerobic and anaerobic respiration in Bacillus
subtilis. The histidine sensor kinase ResE, by functioning as a kinase and a phosphatase for the cognate
response regulator ResD, controls the level of phosphorylated ResD. A high level of phosphorylated ResD is
postulated to cause a dramatic increase in transcription of ResDE-controlled genes under anaerobic condi-
tions. A mutant ResE, which retains autophosphorylation and ResD phosphorylation activities but is defective
in ResD dephosphorylation, allowed partially derepressed aerobic expression of the ResDE-controlled genes.
The result indicates that phosphatase activity of ResE is regulated by oxygen availability and anaerobic
induction of the ResDE regulon is partly due to a reduction of the ResE phosphatase activity during anaer-
obiosis. That elimination of phosphatase activity does not result in complete aerobic derepression suggests that
the ResE kinase activity is also subject to control in response to oxygen limitation.

In two-component signal transduction systems (for reviews,
see references 9 and 29) histidine kinases modulate the activity
of response regulators via phosphorylation. Response regula-
tors have autophosphatase activity, and half-lives for various
phosphorylated response regulators range from seconds to
hours (36). The decay of phosphorylated response regulators is
often stimulated by the cognate sensor kinases that possess
phosphatase activity (for a review, see reference 30). Since the
level of phosphorylation of response regulators is determined
by the sum of kinase, phosphotransferase, and phosphatase
activities, how each of these activities is regulated is a key issue
for understanding the mechanism of signal transduction.
Bacillus subtilis, a gram-positive soil bacterium, can alternate
its respiratory systems depending on the growth conditions.
When nitrate is present in the absence of oxygen, cells undergo
nitrate respiration using nitrate reductase as terminal oxidase
(for a review, see references 18 and 23). Anaerobic nitrate
respiration, as well as aerobic respiration using cytochrome
oxidases, is dependent on the ResDE signal transduction sys-
tem (24, 31). The sensor kinase ResE and the response regu-
lator ResD are required for transcription of resABCDE
(resABC encodes proteins similar to those involved in cyto-
chrome c biogenesis) (31), ctaA (required for cytochrome caa3
oxidase biosynthesis) (31), ctaB (cytochrome caa3 oxidase as-
sembly factor) (13), qcrABC (encoding subunits of menaquin-
ol:cytochrome c oxidoreductase) (31), fnr (encoding anaerobic
transcriptional regulator) (24), nasDEF (nitrite reductase)
(17), hmp (flavohemoglobin) (12), lctE (lactate dehydroge-
nase) (4), and sbo-alb (subtilosin biosynthesis) (20). All
ResDE-controlled genes so far tested are highly induced by
oxygen limitation (21). Recent studies showed that purified
* Corresponding author. Mailing address: Department of Biochem-
istry and Molecular Biology, Oregon Graduate Institute of Science and
Technology, 20000 N.W. Walker Road, Beaverton, OR 97006. Phone:
(503) 748-4078. Fax: (503) 748-1464. E-mail: mnakano@bmb.ogi.edu.
ResD directly interacts with promoter regions of some of these
genes (22, 37).
We have previously shown that pgk-1, a mutation in pgk
(phosphoglycerate kinase gene), suppresses resE but not resD
mutations with respect to anaerobic growth in the presence of
nitrate and to ResDE-dependent gene expression (21). The
pgk-1 mutant displays very low but measurable phosphoglycer-
ate kinase activity compared to the wild-type strain. Accumu-
lation of a glycolytic intermediate, probably 1,3-diphosphoglyc-
erate, was suggested to be responsible for the observed
suppressor effect of pgk-1. However, it remains to be examined
whether 1,3-diphosphoglycerate can donate phosphate directly
to ResD or if a non-cognate kinase is involved in the ResE-
independent ResD phosphorylation. During the study we
found that aerobic expression of the ResDE-controlled genes
was dramatically derepressed in the resE pgk-1 double mutant;
however, the expression in the resE⫹ pgk-1 strain was similar to
that of the wild type, showing much lower expression under
aerobic than anaerobic conditions. One possible explanation
for these results is that ResE has both kinase and phosphatase
activities under aerobic conditions but lacks phosphatase ac-
tivity under anaerobic conditions. In this view, when ResD is
phosphorylated by a pathway independent of ResE, and ResD
phosphate (ResD⬃P) is not dephosphorylated by ResE phos-
phatase, as in case of the resE pgk-1 mutant, a higher level of
ResD⬃P would be attained, leading to robust activation of
ResD-controlled genes. We also proposed that higher expres-
sion of ResDE-controlled genes in the wild-type strain under
anaerobic conditions is likely the result of higher ResD⬃P due
to reduced phosphatase activity of ResE (21). This hypothesis
was tested in this study by creating a mutant ResE that pos-
sesses kinase activity but lacks phosphatase activity.
MATERIALS AND METHODS
B. subtilis strains and plasmids. B. subtilis strains and plasmids used in this
study are listed in Table 1.

1938
VOL. 183, 2001 ResE PHOSPHATASE ACTIVITY IN B. SUBTILIS 1939

TABLE 1. B. subtilis strains and plasmids

Strain or plasmid Relevant feature(s) Source or reference

Strains
JH642 trpC2 pheA1 J. A. Hoch
ZB307A SP␤c2⌬2::Tn917::pSK1 39
LAB2000 trpC2 pheA1 SP␤c2⌬2::Tn917::pML26 12
LAB2234 trpC2 pheA1 ⌬resE::spc 16
LAB2252 trpC2 pheA1 SP␤c2⌬2::Tn917::pMMN288 24
LAB2537 trpC2 pheA1 amyE::pES17 (resA-lacZ) 21
LAB2854 trpC2 pheA1 SP␤c2⌬2::Tn917::pMMN392 17
LAB3162 trpC2 pheA1 SP␤c2⌬2::Tn917::sbo-lacZ 38
ORB3303 resE::neo This study
ORB3304 trpC2 pheA1 resE::neo This study
ORB3331 trpC2 pheA1 amyE::pES17 resE::neo This study
ORB3343 trpC2 pheA1 resE378 amyE::pES17 This study
ORB3362 trpC2 resE378 This study
ORB3370 trpC2 resE378 SP␤c2⌬2::Tn917::pML26 This study
ORB3371 trpC2 resE378 SP␤c2⌬2::Tn917::pMMN392 This study
ORB3372 trpC2 resE378 SP␤c2⌬2::Tn917::pMMN288 This study
ORB3373 trpC2 resE378 SP␤c2⌬2::Tn917::sbo-lacZ This study

Plasmids
pPROEX-1 ColE1 origin with His6, Ampr GIBCO-BRL
pTYB4 ColE1 origin with intein tag, Ampr New England Biolabs
pUC18 ColE1 origin, Ampr 35
pUC19 ColE1 origin, Ampr 35
pMMN424 pTYB4 with resE, Ampr 22
pMMN425 pTYB4 with resE378, Ampr This study
pMMN444 pUC19 with resE, Ampr This study
pMMN446 pPROEX-1 with resE, Ampr This study
pYZ16 pUC18 with resE, Ampr This study
pYZ24 pYZ16 with neo insertion in resE, Ampr Neor This study

Construction of plasmids carrying truncated wild-type resE or resE378 gene.
For production of wild-type and mutant ResE proteins, the IMPACT system
(New England BioLabs) was used which utilizes the inducible self-cleaving intein
tag (3). Plasmid pMMN424, which carries a truncated resE gene in pTYB4, was
constructed previously (22). This plasmid was used to produce and purify a
soluble form of the wild-type ResE protein lacking the N-terminal 195 amino
acids (total amino acids are 589) including two transmembrane regions and the
periplasmic region. The truncated protein was shown to have active kinase
activity (22, 37). The last amino acid, Arg, is also replaced by Gly and Pro in
wild-type and mutant ResE constructs.
Plasmid pMMN425, which was used to purify a truncated form of the mutant
ResE (T378R) protein, was constructed as follows. The upstream fragment of
resE was amplified by PCR using JH642 chromosomal DNA and two oligonu-
cleotides, oMN98-47 (5⬘-CATTCTTTTTATCAACCATGGTCACGTACCCT-
3⬘) and oMN98-49 (5⬘GTCATGAGCTGAGACGACCGATCTCCAT-3⬘). The
downstream fragment of resE was amplified using two oligonucleotides,
oMN98-48 (5⬘-CAGACTCGATTTTACCCGGGTTTTGTCGGAATAT-3⬘) and
oMN98-50 (5⬘-ATGGAGATCGGTCGTCTCAGCTCATGAC-3⬘). oMN98-49
and oMN98-50 are complementary and designed to generate the resE378 muta-
tion. The PCR products were denatured at 94°C for 1 min and were successively
incubated at 65°C for 2 min and 37°C for 1 min. The annealed mixture was
treated with T4 DNA polymerase in the presence of four deoxynucleoside
triphosphates at 37°C for 30 min. The aliquot of the reaction mixture was used
as a template for PCR using oMN98-47 and oMN98-48 to generate the truncated
resE378 gene. The PCR product was digested with NcoI and SmaI and cloned
into pTYB4, which was cleaved with the same enzymes to generate pMMN425.
The inserted DNA was sequenced to verify the desired mutation, as well as the
absence of any extra mutation.
The truncated form of wild-type ResE was also produced as a protein fused to
six-histidine residues (His6). This His6-ResE protein was used to purify ResD⬃P
from His6-ResE by affinity chromatography. Plasmid pMMN424 was digested
with SmaI and NcoI (blunt ended with T4 DNA polymerase), and the released
resE fragment was cloned into pUC19 digested with SmaI. The resultant plasmid
pMMN444 was digested with BamHI and KpnI to release resE, which was
subcloned into pPROEX-1 (GIBCO-BRL) that had been digested with the same
enzymes to generate pMMN446.
Purification of ResD and ResE proteins. ResD, wild-type, and mutant ResE
proteins were overproduced in Escherichia coli ER2566 (New England Biolabs)
as described previously (22, 37). His6-ResE was overproduced in E. coli BL21
carrying pMMN446 and purified using Ni-nitrilotriacetic acid (NTA) resin col-
umn chromatography as recommended by the manufacturer. The His6-ResE
protein has 44 extra amino acid residues including 6 histidines at the N terminus
and 18 extra amino acids at the C terminus of ResE.
Autophosphorylation of ResE. The truncated wild-type and mutant ResE
proteins (60 pmoles) were incubated in 60 ␮l of TEDG buffer (50 mM Tris-HCl,
pH 8.0; 0.5 mM EDTA; 2 mM dithiothreitol [DTT]; 10% glycerol) containing 50
mM KCl, 5 mM MgCl2, and 10 ␮M [␥-32P]ATP (1 Ci/mmol). After incubation
for the indicated periods at room temperature, 10 ␮l of the reaction mixture was
removed and added to 3 ␮l of a sodium dodecyl sulfate (SDS) sample buffer (250
mM Tris-HCl, pH 6.5; 8% SDS; 8% 2-mercaptoethanol; 40% glycerol; 0.05%
bromophenol blue). The proteins were separated by SDS–12% polyacrylamide
gel electrophoresis and analyzed using a PhosphorImager (Molecular Dynam-
ics).
Phosphorylation of ResD by ResE. The wild-type and mutant ResE proteins
(960 pmol) were autophosphorylated with [␥-32P]ATP for 30 min at room tem-
perature as described above. The reaction mixture was applied to a Sephadex
G-75 column equilibrated with the same buffer. The fractions containing the
radioactive ResE, which were free of ATP, were collected. An aliquot of the
fractions was incubated with ResD (300 pmol) in 92 ␮l of TEDG phosphoryla-
tion buffer, and ATP was added to 200 ␮M after 5 min.
Dephosphorylation of ResD⬃P. The His6-ResE protein (0.8 to 1.5 nmol) was
autophosphorylated with [␥-32P]ATP as described above, except that DTT in the
buffer was replaced by 5 mM 2-mercaptoethanol because DTT reduces the Ni
ions of the Ni-NTA resin used for immobilization of His6-ResE. After 30 min at
room temperature, Ni-NTA agarose was mixed into the reaction mixture and
incubated by gently shaking for 15 min. The Ni-NTA resin was collected by
centrifugation and washed with the same buffer to remove unbound His6-ResE
and unincorporated ATP until the radioactive signal in the wash buffer became
constant. An equal amount of ResD was added to the resin and the mixture was
incubated for 10 min at room temperature. The reaction mixture was centri-
fuged, and the supernatant containing ResD⬃P was collected, which was then
applied to a Sephadex G-25 column. For the examination of autophosphatase
1940 NAKANO AND ZHU J. BACTERIOL.

activity, ResD⬃P was incubated in the buffer with or without ATP (500 ␮M) at TABLE 2. Aerobic and anaerobic expression of ResDE-controlled
room temperature. The purified ResD⬃P was also incubated with wild-type and genes
mutant ResE (300 to 500 pmol) in the presence or absence of 500 ␮M ATP or
ADP for 10 min. ␤-Galactosidase activity (Miller unit)a in:
Construction of B. subtilis strains carrying the resE378 mutation. The mutant Fusion DSM 2xYT
resE allele was introduced into B. subtilis as follows. Two fragments carrying the
5⬘-part and 3⬘-part of resE were amplified by using JH642 chromosomal DNA Aerobic Anaerobic Aerobic Anaerobic
and oligonucleotides oMN98-47 and oMN99-57 (5⬘-CTGAAGCATGGGGATC
CGTGTTCTCAG-3⬘), as well as oMN98-48 and oMN99-56 (5⬘-CTGAGAA hmp-lacZ
CACGGATCCCCATGCTTCAG-3⬘). Two complementary oligonucleotides, Wild type 1.7 6,480 3.5 6,550
oMN99-56 and oMN99-57, were designed to create a BamHI site in the resE Mutant 18 8,170 47 5,390
gene. The PCR products, after annealing and being treated with T4 DNA
polymerase as described above, were used as template for the second PCR nasD-lacZ
reaction using oMN98-47 and oMN98-48. The PCR product digested with SmaI Wild type 4.9 540 2.5 546
and NcoI (the end was filled in) was cloned into pUC18 digested with SmaI and Mutant 45 539 16 491
HincII to generate pYZ16. A neomycin-resistant (Neor) cassette isolated from
pDZ792 (8) digested with BamHI and BglII was inserted into the BamHI site of fnr-lacZ
pYZ16 to generate pYZ24. B. subtilis strains JH642 (trpC2 pheA1) and ZB307A Wild type 5.5 78 6.6 199
(trpC2⫹ pheA1⫹) were transformed with pYZ24 that was linearized by ScaI Mutant 47 133 42 224
cleavage, and Neor transformants were selected as ORB3304 and ORB3303,
respectively. The transformants were generated by a double-crossover recombi- resA-lacZ
nation, as was confirmed by PCR analysis. LAB2537 carrying resA-lacZ was Wild type 22 377 16 430
transformed with ORB3304 chromosomal DNA and a chloramphenicol-resistant Mutant 227 593 199 605
(Cmr) Neor transformant was chosen as ORB3331. ORB3304 was transformed
with ORB3331 chromosomal DNA and pMMN425 with selection for Cmr. A sbo-lacZ
Neos Cmr transformant was chosen as ORB3343. Because ORB3331, like Wild type 19 2,720 3.8 2,450
ORB3304, has the Neor cassette in resE, the neomycin sensitivity of ORB3343 is Mutant 440 5,370 66 3,140
indicative of the replacement of resE::neo by the mutant allele of resE in a
Maximal activities during growth are shown. Standard deviations are less
pMMN425. This was further confirmed by sequencing the PCR product obtained than 20% for each value.
by using ORB3343 chromosomal DNA as a template and oMN98-47 and
oMN98-48 as primers. The mutant resE strain without the lacZ fusion was
constructed by transforming ORB3343 with ORB3303 chromosomal DNA. After
selection with trp⫹, a Cms Neos transformant was chosen as ORB3362. ORB3362 regions (22, 37), arguing against the existence of a coactivator
was used for transduction with phage lysates carrying hmp-lacZ (12), nasD-lacZ or a ResD-controlled transcription activator. These results,
(17), fnr-lacZ (24), and sbo-lacZ (38) to construct strains ORB3370, ORB3371, together with studies on a resE suppressor mutant as described
ORB3372, and ORB3373, respectively.
Measurement of ␤-galactosidase activity. B. subtilis cells were grown in liquid
above (21), support the conclusion that the level of phosphor-
2xYT medium (19) with 1% glucose and 0.2% KNO3 or in DS medium (19) with ylation of ResD is the major factor for determining the induc-
1% glucose and 0.2% KNO3 (starting optical density at 600 nm was 0.02). Cells tion of these genes.
were cultured aerobically or anaerobically as previously described (24), and One possibility for why ResD is phosphorylated to higher
samples were taken every 1 h to measure ␤-galactosidase activity as described
levels during anaerobic growth than during aerobic growth is
earlier (15). The maximal activity, which was attained at late exponential growth,
was listed in Table 2. reduced phosphatase activity of ResE under anaerobic condi-
Western blot analysis. Cells were grown as above until late exponential growth tions. This hypothesis was tested in this study by constructing
for anaerobic cultures or T2 (2 h after the onset of the stationary phase) in the kinase⫹ phosphatase⫺ ResE and examining the expression of
case of aerobic cultures. After disruption by French press, cell debris was re- ResDE-controlled genes in the strain producing the mutant
moved by centrifugation (17,000 ⫻ g) for 15 min. The protein concentration in
each sample was determined by using the Bio-Rad assay kit. A total of 20 ␮g of
ResE. If the hypothesis is correct, one could expect dere-
each protein sample was loaded onto SDS–12% polyacrylamide gels. The pro- pressed aerobic ResDE-dependent gene expression in the
teins were detected by Western blot using a chromogenic alkaline phosphatase strain carrying such a mutant ResE. ResE belongs to the EnvZ
substrate, anti-ResE antibody (raised against purified ResE by Josman, LLC, subfamily of sensor kinases (6), and kinase⫹ phosphatase⫺
Napa, Calif.), and secondary goat anti-rabbit alkaline phosphatase conjugate.
EnvZ mutants have been isolated (listed in reference 10). One
such mutant (envZ11) has an amino acid change (Thr247 to
RESULTS AND DISCUSSION Arg) in the vicinity of the conserved His243 residue (the site for
autophosphorylation) (2). Interestingly, an E. coli sensor ki-
Construction of a mutant ResE (T378R). The question of nase CpxA with a change of the equivalent residue Thr to Pro
how the ResDE signal transduction pathway specifically acti- displays gain-of-function phenotype. The CpxA101 mutant
vates either aerobic or anaerobic respiration was addressed in also lacks phosphatase activity for CpxR⬃P (25). Because the
this study. Because ResD and ResE are needed both for aer- corresponding residue (Thr378) is conserved in ResE, we
obic and anaerobic respiration and yet these genes are highly changed the Thr residue to Arg by site-directed mutagenesis
induced under anaerobic conditions in a ResDE-dependent using PCR as described in Materials and Methods. To inves-
manner, several possibilities could be envisioned. One possi- tigate the biochemical activities of wild-type and mutant ResE,
bility is the presence of an unknown regulator that would only the soluble truncated ResE proteins were purified.
be active under anaerobic conditions. This putative regulator Autophosphorylation activities of wild-type and mutant
may be controlled positively by the ResDE system or it may be ResE. ResE, as with other sensor kinases, can undergo auto-
a coactivator of ResD when oxygen is limited. Recent studies phosphorylation in the presence of ATP (22, 37). Purified
indicated that ResD binds to the promoter regions of ctaA, wild-type and mutant (T378R) ResE proteins were examined
resA, hmp, nasD, and fnr, suggesting that ResD activates tran- for autophosphorylation activity. The time course of incorpo-
scription of these genes by interacting with their regulatory ration of the phosphoryl group from [␥-32P]ATP into ResE is
VOL. 183, 2001
FIG. 1. Time course of autophosphorylation of ResE. Wild-type
(A) and mutant (B) ResE proteins (60 pmol) were incubated at room
temperature in 60 ␮l of TEDG buffer (50 mM Tris-HCl, pH 8.0; 0.5
mM EDTA; 2 mM DTT; 10% glycerol) containing 50 mM KCl, 5 mM
MgCl2 and 10 ␮M [␥-32P]ATP (1 Ci/mmol). At the indicated times,
10-␮l samples were taken and analyzed by SDS–12% polyacrylamide
gel electrophoresis and autoradiography. (C) Densitometry scanning
of the gels.
shown in Fig. 1. Both proteins have autophosphorylation ac-
tivity, although the wild-type ResE was phosphorylated at a
higher rate than the mutant. EnvZ11 (2) and CpxA101 (25),
carrying the corresponding mutation, exhibited increased or
diminished autophosphorylation activity, respectively. This re-
sult indicates that the T378R mutation moderately affects the
autophosphorylation activity of ResE.
Phosphorylation and dephosphorylation of ResD by ResE
proteins. To examine transphosphorylation that is indepen-
dent of the autophosphorylation reaction, wild-type and mu-
tant ResE⬃P proteins free of ATP were purified by gel filtra-
tion as described in Materials and Methods. The incubation of
ResE⬃P with ResD resulted in phosphorylation of ResD (Fig.
2). Phosphorylation of ResD either by wild-type or mutant
ResE PHOSPHATASE ACTIVITY IN B. SUBTILIS 1941
ResE occurred quickly and reached a maximum level within
0.5 min. EnvZ catalyzes the dephosphorylation of OmpR⬃P in
the presence of ATP, ADP, or nonhydrolyzable analogs of
ATP (1, 11). The addition of ATP to the reaction mixture also
stimulated the dephosphorylation of ResD⬃P in the reaction
containing wild-type ResE, as shown by the absence of radio-
labeled ResD and ResE after incubation for 5 min at room
temperature (Fig. 2A and C). In contrast, the mutant ResE did
not stimulate ResD⬃P dephosphorylation by the addition of
ATP (Fig. 2B and D). This result indicates that ResE functions
as a phosphatase for ResD⬃P, which is defective in the case of
the T378R mutant.
It has been shown that some two-component regulatory pro-
teins dephosphorylate response regulators in two different
ways: through the phosphatase activity of sensor kinases, which
releases Pi; and through reverse transphosphorylation, which
involves transfer of the phosphoryl group from response reg-
ulators to sensor kinases. Reverse transphosphorylation has
been reported in several two-component regulatory systems,
including NRII-NRI (33), CheA-CheY (28), a kinase⫺ phos-
phatase⫹ EnvZ mutant (5), ArcB-ArcA (7), and PhoR-PhoP in
B. subtilis (27). In an attempt to determine by which process
ResD is dephosphorylated, ResD⬃P was purified from
ResE⬃P by using the His6-ResE construct and Ni-chelate
chromatography (Materials and Methods).
Response regulators have autophosphatase activities, and a
key residue in determining the magnitude of the activity is
amino acid position 56 (in Spo0F), which is adjacent to the site
of phosphorylation of Asp54 (36). Response regulators con-
taining an amino acid residue with a long side chain at the
position equivalent to 56 in Spo0F displayed a low autodephos-
phorylation rate, and those carrying a residue with carboxy-
amide or carboxylate side chain at that position had high de-
phosphorylation rates (36). The corresponding amino acid in
ResD is Met, the same residue present in PhoB, OmpR, and
VanR, which are known to exhibit inefficient autophosphatase
activity (36). Consistent with this observation, our result
showed that autophosphatase activity of ResD is relatively
weak, and the half-life of ResD⬃P was calculated to be ca. 4 h
(Fig. 3A and B). Addition of ATP did not show any significant
effect on autophosphatase activity (data not shown). This long
half-life of ResD⬃P may explain why the phosphatase activity
of ResE is regulated by oxygen tension. When oxygen concen-
tration is increased, the cells need to rapidly decrease the level
of ResD⬃P by activating ResE phosphatase. A similar possi-
bility was suggested in the case of the FixLJ system, where
FixJ⬃P has a relatively long half-life (4 h) (14).
In the presence of ATP or ADP, wild-type ResE stimulates
the dephosphorylation of ResD (Fig. 3C). In contrast, in the
presence of the mutant ResE and ATP, reverse transphospho-
rylation was observed. Reverse transphosphorylation was also
detected in wild-type ResE if EDTA was present. This is in
sharp contrast to the phosphatase activity of the sensor kinases,
which requires Mg2⫹ and was inhibited by the presence of
EDTA. The reverse transphosphorylation from PhoP⬃P to
PhoR does not require Mg2⫹ (27) as in the case of the reaction
from ResD⬃P to ResE.
Effect of the resE378 mutation on transcription of ResDE-
controlled genes. The results using purified ResE proteins in-
dicate that, unlike the wild-type ResE, the mutant ResE lacks
1942 NAKANO AND ZHU J. BACTERIOL.

FIG. 2. Autophosphorylation and dephosphorylation of ResD. 32P-phosphorylated ResE (A) and ResE (T378R) (B) (960 pmol) were purified
and then incubated with purified ResD (300 pmol) in 92 ␮l of TEDG phosphorylation buffer. After incubation for 0.5, 1, 2, and 5 min at room
temperature, 10 ␮l of the reaction was transferred to the SDS buffer. At 5 min, ATP was added to 200 ␮M, and the reaction was continued for
0.5, 1, 2, and 5 min. (C and D) Densitometry scanning of the gels in panels A and B, respectively. Symbols: E, ResE⬃P; F, ResD⬃P.

phosphatase activity, which could result in higher levels of
ResD⬃P. Therefore, we examined whether the resE378 muta-
tion affects ResDE-controlled gene expression in vivo. The
resE gene was replaced by the mutant allele as described in
Materials and Methods. The concentration of wild-type and
mutant ResE proteins in aerobic and anaerobic cultures was
examined by Western analysis using anti-ResE antibody (Fig.
4). Higher levels of ResE proteins were detected in the wild-
type ResE strain grown in 2xYT medium under anaerobic
conditions (360%) than under aerobic conditions (100%). In
contrast, the level of mutant ResE was similar both in aerobic
(450%) and anaerobic cultures (450%) and was as high as the
level of the wild-type ResE protein under anaerobic condi-
tions. The levels of ResE protein in the cells grown in DS
medium were as follows: aerobic wild-type cultures, 100%;
anaerobic wild-type cultures, 410%; aerobic mutant cultures,
200%; and anaerobic mutant cultures, 260% (data not shown).
This indicates that the mutant ResE is indeed produced in
vivo. The higher mutant ResE concentration compared to that
of the wild type during aerobic growth probably reflects the
autoregulation of the resE gene because it is transcribed pri-
marily from the resA operon promoter which is dependent on
ResDE (31).
Various lacZ fusions of ResDE-controlled promoters were
introduced in the wild-type and mutant strains. Expression of
the fusions in both the strains grown aerobically and anaero-
bically in 2xYT medium or in DS medium was examined (Ta-
ble 2). Aerobic expression of all genes was partly derepressed
in the mutant cells grown in 2xYT or DS medium. Aerobic
nasD or fnr expression was six- to ninefold higher in the mutant
than in the wild-type ResE strain. In the case of hmp and resA
expression, aerobic expression was derepressed by 10- to 13-
fold in the mutant strain. The mutation has a more drastic
effect on aerobic sbo expression, which resulted in a 17- to
23-fold increase. In contrast, the anaerobic expression of these
genes was either not affected at all or only slightly increased
(up to twofold) by the mutation.
This result indicates that the resE mutant defective in phos-
phatase activity leads to the partial derepression of the
ResDE-controlled genes under aerobic growth conditions.
However, aerobic expression in the mutant strain is still lower
than anaerobic expression, unlike the situation in the resE
pgk-1 mutant, which showed complete derepression (21). One
possible explanation of the difference is that ResD is phos-
phorylated independently of ResE kinase in the resE pgk-1
mutant, while ResE phosphatase activity is absent, resulting in
higher ResD⬃P levels than those in the resE378 mutant strain,
which has reduced autokinase activity as shown in Fig. 1. An
alternative, but not exclusive possibility, is that the kinase ac-
tivity of ResE, like the phosphatase activity, is also regulated by
oxygen limitation. In aerobic cultures of the resE378 strain
which lacks phosphatase activity, the level of ResD⬃P is high
enough to support a 6- to 20-fold induction compared to cul-
tures of wild-type cells; however, the phosphorylation level
VOL. 183, 2001
FIG. 3. (A and B) Time course of ResD⬃P dephosphorylation. (A)
Purified ResD⬃P was incubated in TEDG buffer containing 50 mM
KCl and 5 mM MgCl2. Samples were transferred at the indicated times
to the SDS buffer and subjected to electrophoresis followed by auto-
radiography. Panel B shows densitometry scanning results of the gel
shown in panel A. (C) Dephosphorylation of ResD⬃P by ResE. Pu-
rified ResD⬃P was incubated at room temperature for 10 min in the
same buffer in the absence (⫺) or in the presence of ResE (wt) or
ResE (T378R) (m) proteins. ATP and ADP were added to 500 ␮M,
and EDTA was added to 10 mM when indicated.
could still be lower compared to anaerobic cultures, the cells of
which not only lack the phosphatase activity but might also
have elevated kinase activity. Autophosphorylation activity of
the sensor kinase FixL of Rhizobium meliloti is stimulated by
low oxygen tension, and the phosphatase activity of FixL⬃P
(but not that of FixL) is depressed under anaerobic conditions
(14). The ResDE regulon could be reciprocally regulated via
kinase and phosphatase activity of ResE according to changes
in the oxygen level, such as the expression of nitrogen fixation
genes in R. meliloti.
ResE is a membrane-associated protein with a type P linker
region (periplasmic signal transducing), which is defined by the
presence of two amphipathic ␣-helices (AS1 and AS2) (34).
The mechanism of signal transduction in this class of sensors
was proposed to involve a conformational change of the
periplasmic region brought about by binding to a signal ligand
(34). The change in conformation is relayed through the cyto-
ResE PHOSPHATASE ACTIVITY IN B. SUBTILIS 1943
FIG. 4. Western analysis of ResE. B. subtilis cells were grown aer-
obically or anaerobically in 2xYT with 1% glucose and 0.2% KNO3. A
total of 20 ␮g of each protein sample was separated by SDS–12%
polyacrylamide gel electrophoresis. After electrophoresis, the proteins
were electrotransferred to a nitrocellulose filter and probed with anti-
ResE antibody. Lanes: M, marker (79.0 kDa); 1, JH642 (wild type)
grown aerobically; 2, JH642 grown anaerobically; 3, ORB3362
(resE378) grown aerobically; 4, ORB3362 grown anaerobically; 5,
LAB2234 (⌬resE) grown aerobically; 6, LAB2234 grown anaerobically.
A cross-reacting band is detected both in resE⫹ and resE strains.
plasmic membrane and causes realignment of the two helices
within the linker region, which, in turn, alters the function of
the C-terminal cytoplasmic domain. It remains to be deter-
mined if the periplasmic region of ResE functions as the sig-
nal-sensing domain and what the signal for ResE is that affects
kinase and/or phosphatase activity. Interestingly, a PAS do-
main, which is known to be an important signaling module for
sensing changes in light, redox potential, and oxygen (32), was
identified in a region adjacent to AS2 (SMART:http://smart
.embl-heidelberg.de/ [26]). The involvement of the PAS do-
main in the redox sensing of ResE remains to be examined.
Future studies are also needed to determine whether the ki-
nase and the phosphatase activities are affected by the same
signal or whether each activity is modulated by distinct signals.
ACKNOWLEDGMENTS
We are grateful to Peter Zuber for valuable discussions and critical
reading of the manuscript. We also thank F. Marion Hulett and Linda
Kenney for helpful advice.
This work was supported by NSF grant MCB9996014.
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