JOURNAL OF BACTERIOLOGY, Dec. 1999, p. 7154–7160 Vol. 181, No.

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Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Bacillus subtilis yckG and yckF Encode Two Key Enzymes of the
Ribulose Monophosphate Pathway Used by Methylotrophs,
and yckH Is Required for Their Expression
HISASHI YASUEDA,* YOSHIO KAWAHARA, AND SHIN-ICHI SUGIMOTO
Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc.,
Kawasaki-ku, Kawasaki-shi, 210-0801, Japan
Received 14 June 1999/Accepted 14 September 1999

The ribulose monophosphate (RuMP) pathway is one of the metabolic pathways for the synthesis of com-
pounds containing carbon-carbon bonds from one-carbon units and is found in many methane- and methanol-
utilizing bacteria, which are known as methylotrophs. The characteristic enzymes of this pathway are 3-hexu-
lose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), neither of which was thought to
exist outside methylotrophs. However, the presumed yckG gene product (YckG) of Bacillus subtilis shows a
primary structure similar to that of methylotroph HPS (F. Kunst et al., Nature 390:249–256, 1997). We have
also investigated the sequence similarity between the yckF gene product (YckF) and methylotroph PHI
(Y. Sakai, R. Mitsui, Y. Katayama, H. Yanase, and N. Kato, FEMS Microbiol. Lett. 176:125–130, 1999) and
found that the yckG and yckF genes of B. subtilis express enzymatic activities of HPS and PHI, respectively. Both
of these activities were concomitantly induced in B. subtilis by formaldehyde, with induction showing depen-
dence on the yckH gene, but were not induced by methanol, formate, or methylamine. Disruption of either gene
caused moderate sensitivity to formaldehyde, suggesting that these enzymes may act as a detoxification system
for formaldehyde in B. subtilis. In conclusion, we found an active yckG (for HPS)-yckF (for PHI) gene structure
(now named hxlA-hxlB) in a nonmethylotroph, B. subtilis, which inherently preserves the RuMP pathway.

The ribulose monophosphate (RuMP) pathway is involved
in formaldehyde fixation in many methylotrophs (11, 15, 17,
22). Typically, the RuMP pathway has two characteristic en-
zymes, 3-hexulose-6-phosphate synthase (HPS) and 6-phos-
pho-3-hexuloisomerase (PHI), and it also shares some en-
zymes with the pentose phosphate pathway and the glycolytic
or Entner-Doudoroff pathway. PHI catalyzes isomerization be-
tween fructose 6-phosphate and hexulose 6-phosphate, which
is synthesized from ribulose 5-phosphate (Ru5P) and formal-
dehyde by HPS. Although HPS and PHI have been detected in
many methylotrophs (3, 12, 18–20, 23, 32, 34), the genes en-
coding these enzymes remain largely unknown. The hps gene,
encoding HPS, was first cloned from an obligate methylotroph,
Methylomonas aminofaciens 77a (44), and cloning of the gene
(rmpB) encoding PHI from M. aminofaciens 77a (35) and a
facultative methylotroph, Mycobacterium gastri MB19 (28), was
recently reported. Neither enzyme was thought to exist outside
methylotrophs, but sequence analysis of the Bacillus subtilis
genome (24, 41) has suggested that yckG may be a homolog of
the gene encoding HPS (44) in M. aminofaciens 77a. The yckG
gene is located between yckF and yckH, at about 32° on the
B. subtilis genome map (24) (Fig. 1a). Here, we present data
showing that a nonmethylotroph, B. subtilis, contains genes
(yckG and yckF) encoding two key enzymes of the RuMP
pathway (HPS and PHI) and that the activities of both en-
zymes can be induced by formaldehyde with dependence on
the yckH gene. These findings indicate the presence of char-
acteristic enzymes from the RuMP pathway in a nonmethyl-
otroph. The significance of detection of these enzymes in B.
* Corresponding author. Mailing address: Fermentation and Biotech-
nology Laboratories, Ajinomoto Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku,
Kawasaki-shi, 210-0801, Japan. Phone: 81-44-245-5972. Fax: 81-44-222-
0129. E-mail: blr_yasueda@te2.ajinomoto.co.jp or Hisashi Yasueda
@Ajinomoto.com..
subtilis is discussed from the aspect of molecular evolution of
the RuMP pathway.
MATERIALS AND METHODS
Bacterial strains, oligonucleotide primers, and plasmids. The wild-type strain
B. subtilis 168 (1) was purchased from the American Type Culture Collection
(Rockville, Md.), and Escherichia coli JM109 (45) was obtained from Takara
Shuzo Co. (Kyoto, Japan). B. subtilis YD101 (yckG-deficient mutant) and YD102
(yckF-deficient mutant) were constructed, as described below, by inserting the
spectinomycin resistance (spc) DNA fragment from pDG1726 (13) (Bacillus
Genetic Stock Center, Columbus, Ohio) into the individual genes of the chro-
mosome of strain 168. Strains YD111 (yckH::spc) and YD112 (yckH::spc) were
also constructed from strain 168 in the same way (Fig. 1a). Strain YD121 (yckH
yckH⫹) contained both the intact yckH gene and the defective one (Fig. 1b).
The potential open reading frame (ORF) of the yckG or yckF gene was cloned
by PCR amplification (29) using the oligonucleotide primer pair BsYck-G1
(5⬘-GAGTATCGATAAAATGGAATTACAGCTTGCATTAGACCTCGT-3⬘;
the translation start codon is underlined, and the restriction enzyme site [ClaI]
for ligation to the vector is italicized) plus BsYck-G6 (5⬘-AATTGTGGATCCC
ATTGAGAATTTCCGCTACGTATTCAGTCG-3⬘; the restriction enzyme site
[BamHI] for ligation to the vector is italicized) or BsYck-F1 (5⬘-AAGCATCG
ATAAAATGAAAACGACTGAATACGTAGCGGAA-3⬘) plus BsYck-F2 (5⬘-
ATCTTGGATCCGGTTGTGTGATGTTATTCAAGGTTTGCG-3⬘). The re-
gion containing the yckG and yckF genes was amplified by PCR using the primer
pair BsYck-G1 plus BsYck-F2. PCR was performed with Takara LA Taq DNA
polymerase (Takara Shuzo) for 28 cycles with purified B. subtilis genomic DNA.
The PCR products were purified with a QIAquick gel extraction kit (Qiagen).
The fragments thus obtained were ligated behind the E. coli trp promoter to
mediate overexpression in E. coli (46). Each amplified DNA fragment was
digested with restriction enzymes ClaI and BamHI, and the fragments were
introduced into the vector to construct the expression vectors pT-Bsb-yckG6,
pT-Bsb-yckF1, and pT-Bsb-yckGF1 for the overexpression of yckG, yckF, and
yckGF, respectively. The fragment containing yckH was amplified by PCR using
primers BsYck-H1 (5⬘-CAATGTTAACGTCAGGCTTTTGCTGGATCACTTC
TGGCA-3⬘; the restriction enzyme site for cloning of the amplified DNA frag-
ment is italicized) and BsYck-H2 (5⬘-GTGTACCGAATTCGTTTTTGTGCAT
CCGTTAAAGGGTA-3⬘). After digestion with HpaI and EcoRI, the yckH
fragment was cloned into pACYC184 (Nippon Gene, Tokyo, Japan) treated with
Bst1107I and EcoRI to construct pAY-Bsb-yckH.
For the purpose of gene disruption, the spc fragment was introduced into the
cloned yckG and yckF genes to construct pT-Bsb-yckG-Sp5 and pT-Bsb-yckF-
Sp6, respectively. The spc gene was also cloned into the PvuII site in the yckH
gene to construct two plasmids, pAY-Bsb-yckH-SpA and pAY-Bsb-yckH-SpB, in

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VOL. 181, 1999 ONE-CARBON COMPOUND METABOLISM IN B. SUBTILIS
FIG. 1. Diagram of the chromosomes of wild-type B. subtilis and its mutants.
(a) Map of the yckF-yckG (phi-hps) and yckH loci showing the protein coding
regions at 32° on the B. subtilis genome (24). Locations and orientations of the
coding regions are indicated by thin black arrows, and potential transcription
terminators, as proposed in the SubtiList data bank (41), are shown. The hps, phi,
and yckH genes are expanded and depicted as thick black arrows below the map.
Insertion of a spc cassette (thick white arrow) leading to disruption of hps in
strain YD101, phi in YD102, or yckH in YD111 or YD112 is shown. (b) Chro-
mosomal structure of strain YD121, which contains the disrupted and intact
yckH genes. Heavy and thin lines depict vector and chromosomal DNA, respec-
tively. Open boxes indicate the yckG, yckF, and yckH genes. The black box shows
the spc gene, which disrupts the yckH gene. Thin arrows show the direction of
transcription.
which the direction of the spc gene was the same as and opposite, respectively,
that of the yckG gene (Fig. 1a).
Construction of B. subtilis mutants. B. subtilis mutants YD101, YD102,
YD111, and YD112 were constructed by the introduction of pT-Bsb-yckG-Sp5,
pT-Bsb-yckF-Sp6, pAY-Bsb-yckH-SpA, and pAY-Bsb-yckH-SpB, respectively,
into the wild-type strain. YD121 was generated from the wild-type strain through
homologous recombination with the yckH region by a Campbell-type event.
Transformation of B. subtilis was performed by the competent-cell method (1),
and spectinomycin (100 ␮g/ml) was used for selection of the transformants.
Preparation of cell extract from E. coli overexpressing the yckG and yckF
genes. E. coli JM109 was transformed with the expression plasmids (36). Trans-
formants were grown at 37°C for 12 h in 5 ml of M9-Casamino Acids medium
(36) supplemented with thiamine-HCl (2 ␮g/ml), indoleacrylic acid (25 ␮l/ml),
and ampicillin (100 ␮l/ml). The cells were harvested by centrifugation and sus-
pended in buffer A (50 mM potassium phosphate [pH 7.6], 1 mM MgCl2). After
centrifugation, the washed cell pellet was resuspended in 1 ml of buffer B (50 mM
potassium phosphate [pH 7.6], 5 mM MgCl2, 1 mM dithiothreitol). A cell extract
was prepared by sonication with a disrupter (Bioruptor; Cosmobio, Tokyo, Ja-
pan) at maximum power with a standard oscillator. The sonicate was clarified by
centrifugation at 15,000 ⫻ g for 20 min at 2°C, and the supernatant thus obtained
was used as the cell extract.
SDS-PAGE analysis. Sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) (14% polyacrylamide) was performed by the method
of Laemmli (25), using a ready-made gel system (TEFCO, Tokyo, Japan). Sam-
ples of cultured cells were collected by centrifugation, and the cell pellets were
suspended in gel loading buffer and heated at 100°C for 5 min. Aliquots of 5 ␮l
were loaded onto an SDS-polyacrylamide gel, which was stained with PhastGel
Blue R (Coomassie blue R-350; Pharmacia AB). Molecular weight standards
consisted of a marker mix consisting of seven recombinant proteins of 15, 25, 35,
50, 75, 100, and 150 kD (Novagen, Madison, Wis.).
Induction of expression of the yckG and yckF genes in B. subtilis. After
overnight growth in Luria-Bertani (LB) medium (36), cultures of wild-type and
mutant B. subtilis were diluted to 1:20 in fresh medium. Cells were cultured at
37°C with shaking until the mid-log phase, and then formaldehyde or another
reagent (methanol, formate, or methylamine) was added as specified in the text.
Other cultures were exposed to the following stresses: heat shock, provided by
culture at 48°C; salt stress, created by adding NaCl at a final concentration of 5%
(wt/vol); and acid shock, provided by adding 0.5 N HCl to reduce the pH of the
culture from 6.8 to 5.2. Incubation was continued for 90 min under these con-
ditions. Next, the cells were harvested, washed, and resuspended in 50 mM
potassium phosphate buffer (pH 7.5) containing 3 mM MgCl2 and 1 mM dithio-
threitol. The cell extract was prepared for assay by sonication and centrifugation
to remove debris.
Assay of HPS and PHI activities. HPS activity was determined as described
previously (3), with some modifications. The reaction mixture (0.95 ml) consisted
7155
of 50 mM potassium phosphate (pH 7.6), 5 mM MgCl2, 5 mM ribose-5-phos-
phate (Ri5P) (Nacalai Tesque, Inc., Kyoto, Japan), 2.5 mM NADP (Sigma, St.
Louis, Mo.), 10 U of phosphoribose isomerase (PRI) (Sigma), 10 U of glucose-
6-phosphate dehydrogenase (Boehringer Mannheim), 10 U of phosphoglucose
isomerase (PGI) (Boehringer Mannheim), 50 U of recombinant PHI purified
from E. coli overproducing M. aminofaciens 77a PHI (35), and the test sample of
cell extract. The mixture was preincubated for 3 min at 37°C to achieve equilib-
rium of Ru5P and Ri5P (catalyzed by PRI), and then the reaction was started by
adding formaldehyde at a final concentration of 5 mM. In the PHI assay, re-
combinant M. aminofaciens 77a HPS (35) was used instead of recombinant PHI,
and both were eliminated from the reaction mixture when the serial reaction of
HPS and PHI enzymes was assayed. Activity was assessed by monitoring the
change in optical density (OD) at 340 nm with NADP as a cofactor, and cor-
rection was made for formaldehyde-independent activity. The protein concen-
tration was determined by the method of Bradford (8) with a Bio-Rad protein
assay kit (Bio-Rad Laboratories, Richmond, Calif.) and bovine serum albumin as
the standard.
Assay of other enzymes. NAD-dependent methanol dehydrogenase (MDH)
and glutathione-dependent formaldehyde dehydrogenase (GS-FDH) were as-
sayed by using the B. subtilis cell extract treated with formaldehyde. MDH ac-
tivity was assayed as described by Arfman et al. (2); GS-FDH activity was assayed
as described by Attwood. (6).
Effect of yckG and yckF mutations on cell growth in the presence of formal-
dehyde or NaCl. B. subtilis 168 (wild type) and two isogenic mutant strains
(YD101 and YD102) were cultured at 30°C with shaking in LB medium con-
taining spectinomycin when necessary. Overnight cultures were inoculated into
warmed fresh LB medium, or into medium containing formaldehyde or NaCl,
and cell growth and lysis profiles were automatically recorded at an OD of 660
nm with a Bio-photorecorder (Advantec, Tokyo, Japan).
13
C NMR analysis of the products obtained with [13C]formaldehyde as the
substrate. A reaction mixture containing 50 mM potassium phosphate (pH 7.6),
5 mM MgCl2, 5 mM Ri5P, 10 U of PRI, and 10 U of PGI was prepared. After
preincubation at 37°C for 10 min to produce Ru5P from Ri5P, cell extract
containing the yckG gene product (YckG) and yckF gene product (YckF) was
added to the mixture. The serial YckG and YckF reaction was started by addition
of [13C]formaldehyde as the substrate. After incubation at 37°C for 20 min,
EDTA was added to the mixture to chelate Mg2⫹, which is required for HPS
activity. The total activity in the mixture was assayed with 13C nuclear magnetic
resonance (NMR) spectroscopy by monitoring the decrease of the resonance
signal for the 13C atom of formaldehyde and the appearance of signals for the C
atoms of fructose 6-phosphate and glucose 6-phosphate, which was yielded by
addition of PGI. In the negative control sample, EDTA was added to the mixture
immediately after addition of the cell extract.
NMR spectra were obtained at 100 MHz with a Fourier transform (FT) NMR
spectrometer (JNM-A400; JEOL, Tokyo, Japan), using 0.6-ml samples contain-
ing 15% D2O in 5-mm-diameter tubes to provide a lock signal. A total of 256
transients were acquired by using 32k data points. Gated proton decoupling was
used, and 45° pulses were applied, with a 2.5-s relaxation delay between pulses to
give a repetition time of 3.9 s. During acquisition, the samples were kept at 25°C.
[13C]formaldehyde (99% 13C atom) was purchased from Cambridge Isotope
Laboratories (Andover, Mass.).
RNA analysis. B. subtilis cells were grown to mid-log phase in LB medium at
37°C. After the addition of formaldehyde at final concentration of 0.5 mM,
culture was continued for 45 min. Then RNA was extracted by using an ISOGEN
kit (Nippongene, Toyama, Japan) and treated with RNase-free DNase (Takara
Shuzo). About 1 ␮g of RNA was mixed with the YGF-R2 oligonucleotide DNA
(5⬘-TTCAGGCCGGTTGTGTGATGC-3⬘), and cDNA was synthesized by using
a reverse transcription (RT) kit (RNA LA PCR kit; Takara Shuzo). YGF-R2
corresponded to the minus strand of DNA just behind the yckF ORF. Then a
20-␮l aliquot of the cDNA mixture was combined with DNA primers YGF-F1
(5⬘-ATGGAATTACAGCTTGCATTAGACC-3⬘) and YGF-R1 (5⬘-TGTGA
TGCTATTCAAGGTTTGC-3⬘), LA Taq DNA polymerase, and PCR buffer
(Takara Shuzo) in a volume of 100 ␮l. RT-PCR was carried out according to the
manufacturer’s instructions. YGF-F1 and YGF-R1 corresponded to the plus
strand of DNA encoding the N-terminal region of YckG and to the minus strand
of DNA encoding the C-terminal region of YckF, respectively. As a negative
control, mixture without RT was used for PCR amplification.
Computer analysis. Nucleotide and amino acid sequences were analyzed with
the Genetyx-Mac computer program (Software Development Co., Tokyo, Ja-
pan). Alignment of deduced amino acid sequences was performed by comparison
with entries in the SWISS-PROT database (release 35.0).
RESULTS
Overproduction and enzyme activity of the yckG and yckF
gene products. To identify the orthologs of the gene encoding
PHI, FASTA searches (30, 31) of the microorganism protein
sequence database were performed, using the amino acid se-
quence of the rmpB gene product (PHI) (35) of M. amino-
faciens 77a as the query sequence. As a result, the primary
7156 YASUEDA ET AL.
FIG. 2. Alignment of amino acid sequences of YckF of B. subtilis (24) and
PHI of M. aminofaciens 77a (35). Identical amino acids and conservative replace-
ments are indicated by asterisks and dots, respectively. Amino acids are num-
bered from the N terminus of each protein.
sequence of the yckF gene product (YckF) in B. subtilis showed
the highest structural similarity with that of PHI (Fig. 2). The
yckF gene is situated five nucleotides downstream of the yckG
gene in the B. subtilis genome (Fig. 1a), and the yckG gene
product (YckG) is known to show structural similarity with
HPS (24). Thus, both putative genes involved in one-carbon
compound metabolism were situated side-by-side in the B. sub-
tilis genome. Because B. subtilis cannot utilize methanol as its
sole carbon source (i.e., it is a nonmethylotroph), the detection
of such genes in this microorganism was interesting with re-
spect to function and evolution.
We first expressed the yckG- and/or yckF ORF in E. coli to
evaluate whether the translated product of each ORF dis-
played HPS or PHI activity. After each corresponding DNA
fragment with the postulated ORF was amplified by PCR, the
fragments were inserted into the expression plasmid and over-
production was stimulated (Fig. 3a). As a result, the yckG gene
yielded a dense protein band with the expected molecular mass
of 23 kDa on SDS-PAGE, and the yckF gene yielded a protein
with a molecular mass of 19 kDa. Furthermore, overexpression
of a DNA fragment containing the yckG and yckF genes in the
intact gene structure led to concomitant overproduction of
both ORF products (Fig. 3a, lane 3), showing that termination
of transcription did not occur between the yckG and yckF
genes in E. coli.
The HPS and PHI activities of cell extracts were examined
by the enzyme assay method. E. coli overexpressing yckG or
yckF showed HPS or PHI activity, respectively (Table 1). To
confirm that formaldehyde was incorporated into pentose
phosphate, we used the 13C NMR technique with [13C]form-
aldehyde. Preincubation of the reaction mixture containing
Ri5P, PRI and other chemicals yielded Ru5P, which is an
acceptor of formaldehyde in the reaction with HPS. Addition
of a cell extract containing YckG and YckF to the mixture
caused the appearance of the corresponding resonance signals
for [13C]fructose-6-phosphate and [13C]glucose-6-phosphate,
along with a decrease of the [13C]formaldehyde signal (Fig.
3b). This showed that the serial reaction catalyzed by both gene
products could progress, because YckG produced hexulose-6-
phosphate from formaldehyde and Ru5P, while YckF and PGI
then created fructose-6-phosphate and glucose-6-phosphate,
respectively. Since the two enzymes were previously thought
to be unique to methylotrophs (11, 15), it was surprising that
J. BACTERIOL.
B. subtilis contained nucleotide sequences that could express
these enzyme activities.
Expression of yckG and yckF genes by B. subtilis. To inves-
tigate whether the yckG and yckF genes intrinsically expressed
any enzyme activity in B. subtilis, we assessed extracts of cells
grown under various culture conditions. The extract of wild-
type cells cultured in LB medium showed negligible HPS or
PHI activity, but both activities were detected after culture in
the presence of formaldehyde (Table 2). When various con-
centrations of formaldehyde were tested, significant HPS and
PHI activity was found in the cell extract at formaldehyde
concentrations of around 0.5 mM. At a concentration of over
1 mM, however, both activities almost disappeared.
Formaldehyde is produced from methanol by methanol de-
hydrogenase and is also oxidized to formate by formaldehyde
dehydrogenase in some methylotrophs (11). Therefore, we ex-
amined whether the synthesis of HPS and PHI could be in-
duced by methanol or formate, but neither enzyme activity was
detected after cells were grown in medium containing metha-
nol or formate (Table 2). On the other hand, since formalde-
hyde is also synthesized by oxidation of methylamine in several
methylotroph species (26), we looked for HPS or PHI activity
in extracts of B. subtilis grown in medium containing methyl-
amine hydrochloride (0.4 or 1.2% [wt/vol], final concentration)
but did not observe the induction of these enzymes. Therefore,
the nonmethylotroph B. subtilis possessed two key enzymes of
the RuMP pathway, and the expression of both enzymes was
induced by formaldehyde.
Analysis of yckG and/or yckF disruption mutants. We next
examined whether the yckG or yckF gene encoded the ob-
served formaldehyde-inducible HPS or PHI activity. We con-
structed a yckG mutant (strain YD101) and a yckF mutant
(strain YD102) by insertional mutagenesis (Fig. 1a) and ana-
lyzed the formaldehyde-inducible HPS or PHI activity of each
strain. After induction with formaldehyde, we detected no sig-
nificant enzyme activity in the YD101 strain and only HPS ac-
tivity in the YD102 strain (Table 2). Thus, we identified YckG
and YckF as the only formaldehyde-inducible HPS and PHI en-
zymes, respectively, and our results also suggested that a polar
effect occurred between the yckG and yckF genes. To confirm
the transcription unit of the two genes, total RNA was isolated
from wild-type B. subtilis cells exposed to formaldehyde and
analyzed by RT-PCR. The presence of mRNA species (of at
least 1.2 kb) spanning the yckG ORF to the yckF ORF (data
not shown) was revealed, suggesting that the two genes (yckG
and yckF) were expressed as a polycistronic mRNA from an
operon.
We also investigated the effect of formaldehyde on the phe-
notype of these mutants. In LB medium, the wild-type strain
and the two mutant strains all showed typical growth and lysis
curves (Fig. 4a). However, after addition of formaldehyde to
the medium, the mutants showed a prolonged lag phase rela-
tive to the growth profile of the wild-type strain, which was
dependent on the concentration of formaldehyde added (Fig.
4). These results indicated that the yckG-yckF system might
endow B. subtilis with the ability to detoxify formaldehyde pro-
duced via endogenous metabolism or environmental changes.
It appeared that the cells were under stress when exposed to
formaldehyde, since growth was arrested for a few hours in its
presence (Fig. 4c and d). To examine whether the increase of
HPS and PHI activities after exposure to formaldehyde was
due to a general stress response rather than to formaldehyde
itself, we assessed the levels of these enzymes in cells grown
under heat (increase from 30 to 48°C), acid (decrease from pH
6.8 to 5.2), and salt (addition of 5% NaCl) stress. We also
examined the growth profiles of wild-type and mutant cells
VOL. 181, 1999 ONE-CARBON COMPOUND METABOLISM IN B. SUBTILIS
FIG. 3. Analysis of the products of yckG and yckF. (a) The yckG and/or yckF
gene was overproduced in E. coli and analyzed by SDS-PAGE. Cells harbor-
ing the expression plasmid were cultured in M9 medium supplemented with
Casamino Acids, thiamine, and metal ions. Indoleacrylic acid was added to
induce the expression of each gene. Aliquots were taken from cells harboring
pT-Bsb-yckG (lane 2), pT-Bsb-yckGF (lane 3), pT-Bsb-yckF (lane 4), and the
control plasmid (lane 5). The single band in lane 1 indicates the position of
purified HPS of M. aminofaciens 77a, and the positions of YckG and YckF are
7157
TABLE 1. HPS and PHI activity in E. coli bearing
an expression plasmid
Sp act (nmol of NADP reduced/min/
Straina mg of protein)b in cell extracts
HPS PHI HPS ⫹ PHIc
E. coli JM109/pT-Bsb-yckG6 3,160 ⬍1 NDd
E. coli JM109/pT-Bsb-yckF1 ⬍1 25,900 ND
E. coli JM109/pT-Bsb-yckGF1 ND ND 3,970
E. coli JM109/pT-Bs-⌬Ncoe ⬍1 ⬍1 ND
a
Cells were grown in modified M9 Casamino Acids medium with indoleacrylic
acid.
b
Mean value from three experiments.
c
Total activity for serial reaction of HPS and PHI.
d
ND, not determined.
e
Negative control.
under salt stress. Enzyme activities were never detected except
after exposure to formaldehyde, but comparison of the growth
profiles of cells exposed to salt stress (5% NaCl) revealed a
slight delay in the growth of both mutants relative to the wild-
type strain. This observation suggests that the yckG or yckF
gene may be involved also in the salt stress response, although
HPS or PHI activity was below the level of detection, as de-
scribed above.
Effect of the yckH gene on expression of the yckG and yckF
genes. Upstream of the yckG-yckF genes, the yckH gene is an
ORF transcribed in the reverse direction from the operon (Fig.
1a). This gene arrangement led us to speculate that yckH might
be a regulator of yckG-yckF expression, by analogy with the
L-arabinose (ara) operon structure (araC and araBAD) (39) in
E. coli. It has been reported that yckH encodes a protein of 120
amino acids (41), but its function remains unclear. Although
the overall amino acid sequence does not share any significant
homology with DNA-binding proteins, a helix-turn-helix motif
that is characteristic of nucleic acid-binding proteins (16) was
indicated at the carboxyl terminus by our prediction of the
secondary structure (data not shown). To examine the effect of
the yckH gene on expression of yckG-yckF, the yckH locus was
inactivated by insertion of an spc gene. We constructed two
mutant strains, YD111 and YD112, in which the directions of
the spc gene were the same as and opposite, respectively, that
of to the yckH gene (Fig. 1a). Although read-through of tran-
scription of the spc gene could proceed into the yckG-yckF
region of the chromosome in YD112, neither of the yckH-dis-
rupted mutants (strains YD111 and YD112) could express de-
tectable HPS and PHI activity when exposed to formaldehyde
(Table 2). We thought that in these mutants, the promoter
element for the yckG-yckF genes might have been destroyed by
spc gene insertion, since the location of the promoter region
was not identified. Therefore, we constructed a new strain,
YD121 (yckH yckH⫹) (Fig. 1b), in which the DNA region just
adjacent to the yckG-yckF locus remained disrupted as in
shown by arrows. The positions of migration of molecular mass standards (SM)
are indicated on both sides of the gel (from top to bottom, 150, 100, 75, 50, 35,
25, and 15 kDa). (b) Formation of glucose-6-phosphate and fructose-6-phos-
phate by the condensation of 13C-labeled formaldehyde and Ru5P and the
subsequent isomerization of sugar-phosphates was analyzed by 13C NMR spec-
trometry as described in the text. The upper profile exhibits the peak of form-
aldehyde (FA) in the test mixture, in which the reaction was immediately stopped
after addition of cell extract containing YckG and YckF; the lower profile
displays the carbon peaks in the reaction mixture after incubation for 20 min at
37°C. The C-1 (␣,␤ anomers) positions of glucose-6-phosphate (G6-P) and fruc-
tose-6-phosphate (F6-P) are indicated.
7158 YASUEDA ET AL. J. BACTERIOL.

TABLE 2. HPS and PHI activities in B. subtilis 168 and deletion anol by MDH in the first stage of the utilization of carbon and
mutants under different growth conditions energy sources by methylotrophs. In B. methanolicus, the syn-
Sp act (nmol of
thesis of HPS is also enhanced by culture with methanol (2),
NADPH produced/ but methanol could not induce HPS or PHI activity in B. sub-
Strain
Growth min/mg of protein)b tilis. Having found that HPS and PHI enzymes exist in B. sub-
conditionsa in cell extracts tilis, we examined the activity of MDH in this microbe. The
HPS PHI HPS ⫹ PHI structure of the cytoplasmic NAD-dependent MDH of B. meth-
anolicus C1 was described by De Vries et al. (9). Using their
168 wild type LB ⬍1 ⬍1 NDc sequence information, we performed a homology search for
LB ⫹ 0.5 mM FA 690 ND 310
LB ⫹ 1.0 mM FA 95 ND ⬍1
the mdh gene in the B. subtilis genome database and found no
LB ⫹ 2.0 mM FA ⬍1 ND ⬍1 significant homologs. In addition, we could not detect NAD-
LB ⫹ 2% (vol/vol) ⬍1 ND ⬍1 dependent MDH activity in cell extracts of B. subtilis cultured
MeOH under various conditions (data not shown). Thus, it appears
LB ⫹ 0.5 mM FM ⬍1 ND ⬍1 that B. subtilis does not possess an NAD-dependent MDH
YD101 (yckG) LB ⫹ 0.5 mM FA ⬍1 ⬍1 ND homolog derived from B. methanolicus. However, it is inter-
YD102 (yckF) LB ⫹ 0.5 mM FA 290 ⬍1 ND
YD111 (yckH) LB ⫹ 0.5 mM FA ⬍1 ⬍1 ⬍1 esting that the possible yeaC gene product of B. subtilis (24, 41)
YD112 (yckH) LB ⫹ 0.5 mM FA ⬍1 ⬍1 ⬍1 displays similarity to the product of mxaR (moxR) required for
YD121 (yckH yckH⫹) LB ⫹ 0.5 mM FA 1,050 ND 200 activity of the pyrrolo-quinoline quinone (PQQ)-linked MDH
a
in a gram-negative methylotroph (42). Besides the methanol
FA, formaldehyde; MeOH, methanol; FM, formate.
b
Mean value from three experiments.
oxidation pathway, formaldehyde is also synthesized from
c
ND, not determined. methylamine in several species of methylotrophs which are
able to utilize methylamine as the sole source of carbon and
energy (21, 26, 27). The yckG-yckF system might be involved in
YD111 but the intact yckH gene was located away from the methylamine metabolism, since some heterotrophs are able to
yckG-yckF region past the intervening sequence of the inte- use methylamine as a nitrogen source, and methylamine, which
grated plasmid. We examined YD121 for formaldehyde-in- is more stable than formaldehyde, is widely distributed in ma-
duced HPS or PHI activity and again detected the induction of rine environment (21). However, no detectable HPS or PHI
both activities. This indicated that the yckH gene is required activity was found in the extract of B. subtilis cells cultured with
for the expression of HPS and PHI activities. methylamine, and methylamine could not support the growth
of B. subtilis as a nitrogen source (data not shown).
This study also indicated a possible role of YckG and YckF
DISCUSSION in the detoxification of formaldehyde. As noted by Attwood
Recently, bioinformatics analysis has suggested that hps gene and Quayle (5), it seems possible that the HPS and PHI system
homologs may be widespread in bacteria (33). We also found is very efficient for trapping free formaldehyde. However, B.
a structural similarity between PHI from a methylotroph (35) subtilis may also equip other detoxification systems for form-
and YckF from B. subtilis and noticed that the hps homolog aldehyde, since our yckG or yckF mutants did not show marked
(yckG) and phi homolog (yckF) were adjacent in the B. subtilis formaldehyde sensitivity. In E. coli, GS-FDH is the primary
genome. In E. coli, it was reported that the SgaH (ORF o216) enzyme that detoxifies formaldehyde (14). Therefore, it is pos-
and SgbH (ORF o220) proteins exhibit sequence similarity to sible that B. subtilis also possesses a reliable detoxification
the HPS of M. aminofaciens (33). Therefore, we assessed the system employing the corresponding enzyme. Indeed, our ho-
enzymatic activity of the sgaH and sgbH gene products from mology search showed that the adhB gene product in B. subtilis
strain W3110 by overexpression of the corresponding genes but is similar to GS-FDH in Methylobacter marinus (40), although
could detect no HPS activity. We also examined HPS activity in we detected no of GS-FDH activity in cell extracts from B.
extracts of E. coli cells cultured under several stresses, includ- subtilis exposed to formaldehyde, as was the case for the meth-
ing formaldehyde exposure, but the activity was below the level anotroph. In addition, we found that both mutants (yckG and
of detection (data not shown). Although HPS activity in E. coli yckF) were slightly more sensitive to salt (NaCl) stress than the
might be induced by a specific effector, it appears from our wild-type strain, although the HPS or PHI activity in cells
results that E. coli does not possess the enzyme. Our homology stressed by salt was too low to be measured. This implies that
search indicated that the E. coli genome does not contain a the promoter responsible for the salt stress response might
homolog of the rmpB (phi) gene (data not shown). These exist in front of both genes or that salt stress could induce the
findings suggested that the E. coli hps homologs may possess a accumulation of formaldehyde during metabolism. The rela-
function other than hexulose phosphate synthesis or that dur- tionship between the salt stress response and the function of
ing its evolution the bacterium has accumulated mutations yckG and yckF remains to be determined.
which impede the expression of HPS activity. According to the The results presented here demonstrate that a nonmethyl-
evolutionary history of prokaryotes based on phylogenetic otroph, B. subtilis, can synthesize two key enzymes (HPS and
analysis (43), B. subtilis is more proximal than E. coli to the PHI) that were previously believed to be specific to methyl-
branch point of the three kingdoms, Archaea, Bacteria, and otrophs employing the RuMP pathway. In B. subtilis, the pen-
Eucarya (7). Therefore, it appears that the presence of a set of tose phosphate pathway has been identified by metabolic flux
hps and phi genes in the genome is critical, and the existence of analysis (37, 38). Therefore, we suggest that B. subtilis pre-
both genes in the B. subtilis genome suggests that the gene serves the RuMP pathway. Although De Wulf assumed the
products were of significant value for this microorganism in its presence of this pathway in B. subtilis on the basis of a prelim-
original environment. inary physiological experiment (10), his conclusion that formic
Expression of the yckG and yckF genes was induced in acid in the culture medium was effectively utilized by B. subtilis
B. subtilis by formaldehyde, similar to what has been found for via the RuMP pathway is not compatible with our results,
two gram-positive methylotrophs, Arthrobacter P1 (27) and Ba- because formic acid could not induce HPS and PHI activities in
cillus methanolicus (4). Formaldehyde is produced from meth- the present study. This discrepancy might be related to differ-
VOL. 181, 1999
FIG. 4. Effect of formaldehyde on the growth of B. subtilis. Overnight cultures of wild-type strain 168 ( ), YD101 (yckG) (•••••), and YD102 (yckF) (– – –)
in LB medium were inoculated into prewarmed LB medium containing formaldehyde at concentrations of 0.0 (a), 0.3 (b), 0.6 (c), and 0.9 (d) mM. Growth and lysis
of cells at 37°C were monitored automatically at an OD of 660 nm.
ences in the B. subtilis strains used. Either way, the existence of
two key enzymes was substantially established by our research.
The organization of divergent transcription between the two
transcription terminators in the B. subtilis genome suggested
that yckH may be a regulator gene (24). The putative gene
product of yckH is estimated to be a polypeptide composed of
120 amino acids (41). However, our disruption experiment
indicated that the yckH gene is required for the expression of
HPS and PHI activities, and it appears that yckH (now named
hxlR) may positively regulate yckG-yckF gene expression
through DNA binding either directly or indirectly after stimu-
lation by formaldehyde. We also suggest that yckG and yckF
(now named hxlA and hxlB, respectively) are organized into an
operon structure, based on the following data: (i) the existence
of mRNA species covering the hxlA and hxlB ORFs, (ii) the
polar effect between hxlA and hxlB genes, (iii) the simulta-
neous expression of the two genes in response to an inducer,
(iv) the lack of hxlB expression concomitant with abolition of
hxlA expression in an hxlR (yckH)-deficient mutant, and (v) the
gene organization of hxlA-hxlB followed by a typical transcrip-
tional terminator. Recently, it was shown that M. gastri MB19
contains a phi (rmpB)-hps (rmpA) operon (28), although the
ONE-CARBON COMPOUND METABOLISM IN B. SUBTILIS 7159
hps (rmpA) and phi (rmpB) genes of M. aminofaciens 77a are
separated by an insertion element (35). We found that the hps
(hxlA)-phi (hxlB) gene organization was conserved as a set of
both genes even in a nonmethylotroph. Curiously, the genes of
B. subtilis and M. gastri were arranged in reverse order. There-
fore, more detailed bioinformatic analysis of the conserved
features and differences in gene organization among three
types of microbes (facultative methylotrophs, obligate methy-
lotrophs, and nonmethylotrophs) may provide insight into the
evolution of the RuMP pathway.
ACKNOWLEDGMENTS
We gratefully acknowledge N. Kato, Y. Sakai, and R. Mitsui (Kyoto
University) for the generous gift of the enzyme samples (HPS and PHI
from M. aminofaciens 77a) and for helpful discussion; E. Suzuki and N.
Ootu for NMR analysis; and K. Sato, M. Ooba, and K. Kobayashi for
technical support.
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