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Infection and Immunity, August 2000, p. 4773-4777, Vol. 68, No. 8
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of the Streptococcus
mutans Pyruvate Formate-Lyase (PFL)-Activating Enzyme Gene by
Complementary Reconstitution of the In Vitro PFL-Reactivating
System
Yasuhito
Yamamoto,1,*
Yutaka
Sato,1,2
Shoko
Takahashi-Abbe,3
Nobuhiro
Takahashi,3 and
Harutoshi
Kizaki1,2
Department of
Biochemistry1 and Oral Health Science
Center,2 Tokyo Dental College, Mihama-ku,
Chiba City 261-8502, and Department of Oral Biochemistry,
School of Dentistry, Tohoku University, Aoba-ku, Sendai City
980-8575,3 Japan
Received 10 January 2000/Returned for modification 6 March
2000/Accepted 26 April 2000
 |
ABSTRACT |
The act gene was identified and an act
mutant as well as the pfl mutant was constructed in
Streptococcus mutans. Pyruvate formate-lyase (PFL) activity
was regenerated with the mixture of the respective cell extracts from
these mutants by complementary reconstitution of the in vitro
reactivating system. The S. mutans act gene encoded the
sole enzyme able to activate the PFL protein in this organism.
| |
TEXT |
Streptococcus mutans has
been implicated as an important microbial agent in human dental caries
(13). The regulation of acid production in this organism
inhabiting dental plaque on tooth surfaces has been of particular
interest because the resulting acid fermentation products from dietary
sugars can demineralize the enamel tooth surfaces, resulting in dental caries.
In the natural environment of this organism, such as the interior of
dental plaque, where anaerobic conditions exist, pyruvate formate-lyase
(PFL) may play a more important role than lactate dehydrogenase (LDH)
in pyruvate metabolism as a part of ATP synthesis and NAD+
and/or NADH recycling (2, 6, 27, 28). In addition, PFL of
this organism is essential for anaerobic metabolism of sugar alcohols
(e.g., sorbitol and mannitol) used as sweeteners for dental caries
prevention (27). S. mutans produces various fermentation end products, including formate, acetate, and ethanol as
well as lactic acid (27). Investigations of the
enzymatically regulatory mechanisms controlling the production of
fermentation end products have revealed that PFL plays a central role
in fermentation in S. mutans (1, 2, 27).
Therefore, this regulatory mechanism should be elucidated at the
molecular level in this organism.
The Escherichia coli PFL has been identified as an enzyme
containing a glycyl radical in its active structure (7, 8, 16, 22,
24) and can be converted from the reversibly inactive form to the
active form by the PFL-activating enzyme, PFL activase (9, 10, 16,
23). The S. mutans PFL was also reported to be
extremely dioxygen sensitive (2, 20, 29). We have cloned the
S. mutans pfl gene encoding PFL (30), and the
deduced amino acid sequence contains the putative glycyl radical at a similar position relative to the E. coli enzyme. Therefore,
it is possible that S. mutans PFL could be activated by PFL
activase or an equivalent enzyme.
In the present investigation, an act gene encoding PFL
activase from S. mutans was cloned by PCR amplification. We
describe the sequence analysis of the S. mutans act gene and
present evidence that it is involved in the regulation of PFL.
Cloning and sequence analysis of the S. mutans act
gene.
To isolate the act gene of S. mutans,
we performed PCR amplification of an act internal region
from S. mutans GS-5IS3 (30) chromosomal DNA. A
425-bp fragment was amplified by PCR using synthesized oligonucleotide
primer sets consisting of act5'2 (5' TATTGCCATAATCCGGAYACNTGG 3'
[forward]) and act3'3 (5' GGAACCAAGACATRKCKDATCCA 3' [reverse])
based upon the sequence of the act sequence of E. coli (14), Clostridium pasteurianum
(25), and a putative act sequence of
Streptococcus pyogenes (The University of Oklahoma's Advanced Center for Genome Technology [http://
www.genome.ou.edu/strep.html]). The three nucleotides at the
3' ends of act5'2 and act3'3 were designed according to the unique
tryptophan codon UGG. The nucleotide sequence of the 425-bp amplicon
was determined and indicated that this amplicon was very likely a part
of the act gene of S. mutans because of its
similarity to the act genes from other organisms. To obtain
the fragments upstream and downstream from the amplified act
internal region, inverse PCR was carried out by using genome walking
libraries of this organism. The nucleotide sequence of the 1.5-kb
ScaI-HindIII region encompassing the entire
S. mutans act gene region was then determined (accession
number AB018417 [http://www.genome.ou.edu/strep.html]) by an
automated DNA sequencer (model 373S; PE Biosystems, Foster City,
Calif.). Nucleotide sequence analysis was carried out with the
DNASIS-Mac program (Hitachi Software Engineering, Yokohama, Japan). The
789-bp open reading frame (ORF) encodes a 263-amino-acid protein with a
calculated molecular weight of 30,148. A potential ribosome-binding
sequence (AGGA) and a promoter-like sequence
(TAGTCT-N18-TATAAT) could be identified immediately
upstream from the putative initiation codon of the act gene.
An inverted repeat sequence characteristic of transcription terminator
could also be detected 11 bp downstream from the termination codon of
the act gene. The formation of a putative stem-loop
structure in this region of the mRNA corresponds to a free-energy
change of 8.4 kJ per mol. Although random mutagenesis to isolate the
act gene was initially attempted as previously reported for
isolation of the pfl gene (30), act
mutants were not isolated by this procedure. In fact, only one
Sau3AI fragment capable of gene inactivation by this
mutagenesis was detected, corresponding to a size of 156 bp based upon
subsequent sequencing of the S. mutans act region. This size
may have been too small for multiple integrational inactivations.
While we were conducting this study, the nucleotide sequences of 10 genes in the locus including the PFL activase gene (pflC) from S. mutans strain LT11 appeared in the GenBank
nucleotide sequence database with the accession number AF051356,
deposited by D. A. Boyd et al. (University of Manitoba, Winnipeg,
Canada) (unpublished data). They reported that the pflC gene
was located in the downstream region of the abcX gene, which
encodes an ATP-binding cassette transporter protein. Our nucleotide
sequence data of the act gene matched that of the
pflC gene except for the 874th nucleotide, which was a T in
our sequence. The amino acid sequence deduced from the act
gene exhibited perfect consistency with that from the pflC gene.
All of the characterized bacterial
act gene homologs are
found downstream of the
pfl gene homologs. For example, the
E. coli and
C. pasteurianum act genes are located
downstream of their
pfl genes (
14,
16,
25).
However, we did not detect any sequences
homologous to the
act gene in a similar region of the
S. mutans chromosome (
30). The
act homologous sequence was
not reported
either upstream or downstream from the
pfl gene
in
Lactococcus lactis or
Streptococcus bovis
(
3,
4). The
pfl gene was located
on the
NotI A fragment (433 kb) on the
S. mutans
chromosome (
5).
Meanwhile, by Southern hybridization
analysis following pulsed-field
electrophoresis we confirmed that the
act probe hybridized with
the
NotI E fragment
(223 kb) (data not shown). These results indicated
that the
act gene was not closely linked to the
pfl gene
on the
S. mutans chromosome.
The amino acid sequences deduced from the
act genes
(
S. mutans and
E. coli) and a putative
act gene (
S. pyogenes) were aligned
for
comparison (Fig.
1) (
14); The
University of Oklahoma's Advanced
Center for Genome Technology
[
http://www.genome.ou.edu/strep.html]).
These three
sequences have 42.1% identical and 79.3% conserved
amino acid
residues overall and have a conserved three-cysteine
cluster, Cys-37,
Cys-41, and Cys-44 (according to residue numbering
in the
S. mutans sequence), which corresponds to the catalytic
site of the
enzyme in
E. coli (
11,
26).
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FIG. 1.
Multiple alignment of PFL activase sequences. The
S. mutans PFL activase sequence was aligned with the
S. pyogenes and E. coli sequences (allowing gaps
[hyphens]). Identical and similar amino acid residues are indicated
by asterisks and colons, respectively, in the consensus sequence. The
region of the three-cysteine cluster, which is the catalytic site in
the E. coli enzyme, is boxed.
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|
Another ORF, designated
act-5'orf, was detected on the same
strand immediately upstream from the promoter-like sequence of
the
act gene. The putative transcription terminator sequence of
the
act-5'orf overlapped with the promoter-like sequence of
the
act gene. A part of this ORF exhibited similarity to a
hemolysin
gene (
tlyC) from
Haemophilis
influenzae.
Characterization of the S. mutans act gene.
To
confirm the function of the act gene, we constructed the
S. mutans act mutant (YASC9YK2) by insertional inactivation
with a suicide vector containing the tetracycline-resistant gene,
following the transformation procedure routinely used in this
laboratory (15, 30). The S. mutans act-5'orf
mutant (YASCXYGEmr1) was also constructed by the same
procedure with the erythromycin-resistant gene to determine whether or
not the protein coded by act-5'orf gene is involved in
pyruvate metabolism or its regulation. The predicted insertion of the
vectors into the chromosome of parental strain (S. mutans
GS-5IS3) was confirmed by Southern hybridization analysis (data not
shown). The PFL and LDH activities with cell extracts from the parental
strain and its mutants (act mutant:YASC9YK2; pfl
mutant:SAKC5Y2C1 (30); act-5'orf
mutant:YASCXYGEmr1) were spectrophotometrically determined
by recording the change in absorbance at 340 nm. The parental strain
was grown in medium D87M containing 1% glucose as carbon source
(D87M-Glu). The act and pfl mutants and the
act-5'orf mutant were grown in D87M-Glu containing 3 µg of
tetracycline per ml and 10 µg of erythromycin per ml, respectively.
Preparation of cell extracts from these cells at the earlier stages of
the exponential growth phase and enzyme assays was performed throughout
under strictly anaerobic conditions in a specially designed anaerobic
chamber as described previously (2, 20, 21). This was
necessary since the S. mutans PFL is very likely a glycyl
radical enzyme that can be converted from the reversibly inactive form
(R form) to the active form (A form) by PFL activase as indicated in
Fig. 2 and also because the A form has
been reported to be extremely dioxygen sensitive (2, 20, 27,
29). The protein concentrations in the cell extracts were
determined by the biuret method (12). The PFL activities
were not detected in the cell extracts from either act or
pfl mutants, while the activities (2.11 ± 0.44 and 2.12 µmol/min/mg of protein) were almost identical in the extracts from the parental strain and the act-5'orf mutant (Table
1). Moreover, we did not detect formate
as a fermentation end product in supernatants from the anaerobic
cultures of both the act and pfl mutants by
assaying as described previously (data not shown) (20). The
LDH activities of the mutants were similar to that of the parental
strain (Table 1). Inactivation of the act-5'orf gene did not
induce any phenotypic changes concerning pyruvate metabolism, e.g.,
formate production, lactate production, and growth with various sugars.
These results apparently indicate that the act mutant was
not able to generate the A form of PFL and the act-5'orf is
not involved in pyruvate metabolism.
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FIG. 2.
Proposed model for interconversion of the reversibly
inactive, active, and irreversibly inactive forms of the S. mutans PFL (21, 27).
|
|
To detect PFL activating activity of the protein encoded by the
act gene, we employed complementary reconstitution of
the
in vitro PFL reactivating system with mixtures of the
respective
cell extracts from the
S. mutans act and
pfl mutants. The
act mutant would not be expected
to express the A form of PFL but
would retain the intact PFL protein as
the R form. In addition,
the
pfl mutant should retain an
enzyme functioning as the PFL
activase. The R form of PFL was
reactivated to the A form by the
PFL activase in the cell extracts
following incubation with 0.6
mM
S-adenosyl-
L-methionine, 10 mM sodium pyruvate,
2.85 mM dithiothreitol,
2 mM
Fe(SO
4)
2(NH
4)
2, and 1 mM methylviologen in 100 mM potassium
phosphate buffer (pH 6.5)
for 90 min at 30°C under strictly anaerobic
conditions (Fig.
2)
(
20,
21). To convert the A form of PFL
in the cell extracts
from parental strain GS-5IS3 to the R form,
the extracts were incubated
for 90 min at 35°C followed by storage
at 4°C for several days
under strictly anaerobic conditions (Fig.
2) (
20,
21,
27).
These extracts derived from parental strain
containing the R form of
PFL converted from the A form of PFL
were used as a control experiment
for complementary reconstitution
of the in vitro PFL reactivating
system with the
pfl and
act mutants,
and
reactivation of PFL by this reactivating system was determined
with the
parental strain (Fig.
3A). The PFL
activity decreased
to approximately 30% (0.64 µmol/min/mg of
protein) of the value
before inactivation (2.11 ± 0.44 µmol/min/mg of protein) and increased
with the incubation time in
reactivating mixtures containing the
R form of PFL from these extracts
(Table
1 and Fig.
3A). The
maximum reactivation (1.12 µmol/min/mg of
protein) was observed
after 90 min of incubation, and the activity was
approximately
1.8 times that before incubation. However, this activity
value
was not fully restored to that before inactivation by this in
vitro reactivating system using methylviologen as a reductant.
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FIG. 3.
Measurement of the reactivated PFL activities. Shown are
results of reactivation of PFL with the cell extracts from the parental
strain (S. mutans GS-5IS3) (A) and with the mixture of
both cell extracts from the act and pfl mutants
(B). The PFL activity was measured at 30°C, and bars represent means
of enzyme activities resulting from two independent experiments. ND,
not detected; air, reactivating mixture was exposed to air for 5 min
after the 120-min incubation.
|
|
We expected that cell extracts prepared from the
act and
pfl mutants would complement each other for PFL activation,
although
both extracts did not independently exhibit PFL activities.
The
detected enzyme activities in the mixture of both extracts
increased
hyperbolically with incubation (Fig.
3B). To confirm that
this
activity was actually derived from the A form of PFL, the
reactivating
mixture was exposed to the air. The A form of PFL is
irreversibly
inactivated due to a cleavage of its peptide (I form) at
the glycyl
radical by dioxygen under aerobic conditions
(
24). In addition,
other
S. mutans enzymes
converting pyruvate into acetyl coenzyme
A such as pyruvate
dehydrogenase complex are not inactivated by
dioxygen. The restored
enzyme activity completely disappeared
when the reactivating mixture,
after the 120-min incubation, was
exposed to the air for 5 min (Fig.
3B). This complete abrogation
of the activity apparently indicates that
our cloned
act gene
encoded PFL activase. The highest
reactivated PFL activity (1.01
µmol/min/mg of protein) observed after
the 120-min incubation
was approximately 90% of that detected in the
control experiment
with the extract from the parental strain (Fig.
3B).
Another dioxygen-sensitive anaerobic ribonucleotide reductase (ARNR)
which catalyzes conversion of ribonucleotides to 2' deoxy
ribonucleotides was reported to be a glycyl radical enzyme in
E. coli (
18,
19).
S. mutans ARNR utilizes
ribonucleoside triphosphate
as a substrate and mainly functions under
strictly anaerobic conditions
(N. Okada, personal communication). The
active form of
E. coli ARNR is similar to that of PFL and
undergoes oxygenolytic cleavage
of the polypeptide chain in vitro at
the site of the glycyl radical
(
16,
18). The ARNR-activating
enzyme exhibited significant
similarity to PFL activase at the primary
sequence level in
E. coli (
16,
17). Our finding
that the
act mutant did not exhibit
PFL activity under
anaerobic conditions excluded the possibility
that the ARNR-activating
enzyme of
S. mutans might complement
the function of the PFL
activase. Together with the results indicated
in Fig.
3, these results
suggest that PFL activase encoded by
the
act gene was the
sole enzyme able to activate PFL in
S. mutans,
although we
cannot exclude the possibility that the PFL activase
may activate
ARNR.
Nucleotide sequence accession number. The
act
nucleotide sequence data appear in the DDBJ, EMBL, and GenBank
nucleotide
sequence databases with the accession number
AB018417.
| |
ACKNOWLEDGMENTS |
We thank T. Yamada (School of Dentistry, Tohoku University) and
H. K. Kuramitsu (State University of New York at Buffalo) for
critical reading of the manuscript, Y. Yamashita (School of Dentistry,
Kyushu University) and M. Kitamura (Himeji Institute of Technology) for
their helpful comments, and Y. Mito (School of Dentistry, Tohoku
University) and A. Gokan (Tokyo Dental College) for their kind assistance.
This investigation was supported in part by grants-in-aid for
Encouragement of Young Scientists (no. 07771676, 08771623, and 09771547 to Y. Yamamoto) from the Japanese Ministry of Education, Science,
Culture, and Sports and a grant-in-aid for Research supported by Tokyo
Dental College.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Tokyo Dental College, Masago 1-2-2, Mihama-ku, Chiba City 261-8502, Japan. Phone: 81-43-270-3750. Fax: 81-43-270-3752. E-mail: yayamamo{at}tdc.ac.jp.
Editor:
V. J. DiRita
| |
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0019-9567/00/$04.00+0
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