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Identification of a Mutation in the Bacillus
subtilis S-Adenosylmethionine Synthetase
Gene That Results in Derepression of
S-Box Gene Expression
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Brooke A. McDaniel, Frank J. Grundy, Vineeta P. Kurlekar,
Jerneja Tomsic and Tina M. Henkin
J. Bacteriol. 2006, 188(10):3674. DOI:
10.1128/JB.188.10.3674-3681.2006.
JOURNAL OF BACTERIOLOGY, May 2006, p. 3674–3681
0021-9193/06/$08.00⫹0 doi:10.1128/JB.188.10.3674–3681.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 10
Identification of a Mutation in the Bacillus subtilis
S-Adenosylmethionine Synthetase Gene That Results
in Derepression of S-Box Gene Expression
Brooke A. McDaniel, Frank J. Grundy, Vineeta P. Kurlekar, Jerneja Tomsic, and Tina M. Henkin*
Department of Microbiology, The Ohio State University, Columbus, Ohio 43210
Genes in the S-box family are regulated by binding of S-adenosylmethionine (SAM) to the 5ⴕ region of the
mRNA of the regulated gene. SAM binding was previously shown to promote a rearrangement of the RNA
structure that results in premature termination of transcription in vitro and repression of expression of the
downstream coding sequence. The S-box RNA element therefore acts as a SAM-binding riboswitch in vitro. In
an effort to identify factors other than SAM that could be involved in the S-box regulatory mechanism in vivo,
we searched for trans-acting mutations in Bacillus subtilis that act to disrupt repression of S-box gene expression during growth under conditions where SAM pools are elevated. We identified a single mutant that proved
to have one nucleotide substitution in the metK gene, encoding SAM synthetase. This mutation, designated
metK10, resulted in a 15-fold decrease in SAM synthetase activity and a 4-fold decrease in SAM concentration
in vivo. The metK10 mutation specifically affected S-box gene expression, and the increase in expression under
repressing conditions was dependent on the presence of a functional transcriptional antiterminator element.
The observation that the mutation identified in this search affects SAM production supports the model that the
S-box RNAs directly monitor SAM in vivo, without a requirement for additional factors.
transcription termination system is sufficient to promote termination of transcription at S-box leader region terminators in
the absence of cellular factors other than RNA polymerase
(RNAP) (4, 20, 39). The S-box RNAs were shown to interact
directly and specifically with SAM, and binding of SAM results
in a structural rearrangement that includes stabilization of
helix 1. Mutations that result in loss of repression during
growth in the presence of methionine also result in loss of
SAM-directed transcription termination and SAM binding in
vitro (20, 21). Overexpression of SAM synthetase in vivo results in increased repression of S-box gene expression, supporting the model that SAM is the effector in vivo (3, 20). These
results suggested that S-box RNAs directly sense SAM and are
therefore members of the family of RNA elements termed
“riboswitches,” which monitor regulatory signals without a requirement for accessory factors such as RNA-binding proteins
or ribosomes (12, 23, 33).
While SAM is sufficient for RNA binding and transcription
termination in vitro, it was not clear whether other factors
might be involved in S-box gene regulation in vivo. We therefore attempted to identify trans-acting mutations that would
lead to loss of repression of S-box gene expression during
growth in the presence of methionine. We predicted that we
could isolate mutations in genes that affect the molecular effector (SAM) or in genes involved in the folding of the leader
RNA, the SAM/leader RNA interaction, or transcription termination in response to SAM. We identified a single transacting mutation that specifically affected S-box gene expression, and the mutation was identified as a nucleotide
substitution in the coding region of metK, the gene encoding
SAM synthetase. This mutation was shown to result in decreased SAM synthetase activity and reduced SAM pools during growth in the presence of methionine. These results are
The S-box regulatory system is used in low-G⫹C grampositive organisms, including members of the Bacillus/Clostridium/Staphylococcus group, to regulate expression of genes involved in biosynthesis and transport of methionine and
S-adenosylmethionine (SAM) (9–11, 29). Genes in the S-box
family exhibit a pattern of conserved sequence and structural
elements in the 5⬘ region of the mRNA, upstream of the start
of the regulated coding sequence(s). These conserved elements include an intrinsic terminator and a competing antiterminator that can sequester sequences that otherwise form the
5⬘ portion of the terminator helix; residues in the 5⬘ region of
the antiterminator can also pair with sequences located further
upstream, and this pairing results in formation of a structure
(the anti-antiterminator) that sequesters sequences necessary
for formation of the antiterminator. The anti-antiterminator
helix (helix 1) (Fig. 1) is located at the base of a complex
structure comprised of helices 1 to 4. Genetic analyses of S-box
leader RNAs supported the model that formation of the antiantiterminator and transcription termination occur during growth
under conditions where methionine is abundant, while starvation
for methionine results in destabilization of the anti-antiterminator, allowing antiterminator formation and readthrough of the
transcription termination site (9). Mutational analysis also suggested that the helix 1 to 4 region is likely to be the target for
binding of a negative regulatory factor or factors (9, 38) and that
pairing between residues in the terminal loop of helix 2 and the
region between helices 3 and 4 is important for termination during growth in high methionine (21).
Recent studies revealed that addition of SAM to an in vitro
* Corresponding author. Mailing address: Department of Microbiology, The Ohio State University, 484 W. 12th Ave., Columbus, OH
43210. Phone: (614) 688-3831. Fax: (614) 292-8120. E-mail: henkin.3
@osu.edu.
3674
Downloaded from http://jb.asm.org/ on February 28, 2014 by PENN STATE UNIV
Received 7 October 2005/Accepted 25 February 2006
B. SUBTILIS SAM SYNTHETASE MUTANT
VOL. 188, 2006
therefore consistent with the model that SAM is the key effector both in vivo and in vitro.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The Bacillus subtilis strains used in
this study were 168 (trpC2); BR151 (lys-3 metB10 trpC2); BR151MA (lys-3
trpC2); ZB307Spc (SP␤c2del2::Tn917erm/spc::pSK10⌬6), a derivative of strain
ZB307A (SP␤c2del2::Tn917::pSK10⌬6) (41) in which the Tn917 erm gene is
replaced by a spectinomycin resistance cassette (31); and IS56B (lys-3 metB relA1
sra-3 strA), a derivative of strain IS56 (lys-3 relA1 trpC2) (30) containing a
mutation (sra-3) that suppresses the partial methionine auxotrophy conferred by
the relA1 allele (unpublished results). Strains containing lacZ fusions are designated with the original strain name followed by the description of the fusion (e.g.,
IS56B::yitJ-lacZ). B. subtilis mapping strains 1A627 through 1A645 (34) were
obtained from the Bacillus Genetic Stock Center (Ohio State University), and
generalized transducing phage PBS-1 was obtained from P. Setlow (University of
Connecticut Health Center, Farmington). B. subtilis strains were grown on tryptose blood agar base medium (TBAB; Difco), Spizizen minimal medium (2),
2XYT broth (22), or A3 medium (antibiotic medium 3; Difco). Antibiotics were
added as indicated at the following concentrations: chloramphenicol, 5 ␮g/ml;
erythromycin, 1 ␮g/ml; lincomycin, 25 ␮g/ml; neomycin, 5 ␮g/ml; spectinomycin,
25 ␮g/ml. X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside; Gold Biotechnologies) was used at 40 ␮g/ml as an indicator of ␤-galactosidase activity. All
growth was at 37°C.
Genetic techniques. Transformation of B. subtilis was carried out as described
previously (14). Chromosomal DNA was prepared using the DNeasy tissue kit
(QIAGEN). Wizard columns (Promega) were used for plasmid preparations.
Oligonucleotide primers were purchased from Integrated DNA Technologies
(Coralville, IA). Restriction endonucleases and DNA-modifying enzymes were
purchased from New England Biolabs and used as described by the manufacturer. Plasmid pGEM7Zf(⫹) (Promega) was used for cloning of metK gene
fragments. Mutations were identified by DNA sequencing (Genewiz Inc., North
Brunswick, NJ).
Transcriptional fusions were generated in plasmid pFG328 (13), which contains a cat gene conferring resistance to chloramphenicol, and integrated in
single copy into the B. subtilis chromosome by transformation of strain
ZB307Spc, which contains a variant of specialized transducing phage SP␤ that
carries a spectinomycin resistance cassette. Strains containing lacZ fusions were
grown in the presence of chloramphenicol or spectinomycin. Transformation of
strains containing lacZ fusion constructs with plasmid pFGneo, a derivative of
plasmid pFG328 that contains a neomycin resistance gene cassette in place of the
cat gene, was used to replace both the cat gene and the adjacent promoter/leader
region directing lacZ expression with a neomycin resistance cassette, by using
flanking plasmid sequences to direct homologous recombination.
Methanesulfonic acid ethyl ester (EMS; Sigma) was used for generalized
mutagenesis. Cells were grown in 2XYT medium to early exponential growth
phase. EMS (1% final concentration) was added, and cells were incubated with
shaking for an additional 100 min at 37°C, pelleted by centrifugation, and washed
twice with Davis salts [0.7% K2HPO4, 0.3% KH2PO4, 0.1% (NH4)2SO4, and 0.005%
MgSO4 · 7H2O]. The cells were then resuspended in 2XYT medium and incubated
with shaking for 3 h at 37°C before addition of 10% glycerol and storage at ⫺70°C.
EMS-mutagenized cells were diluted in 2XYT medium, grown for 30 min, and
plated on TBAB containing X-Gal as an indicator of lacZ expression.
Preparation of PBS-1 transducing lysates and transductions were carried out as
described previously (14). PBS-1 transducing lysates were generated from 19 B.
subtilis mapping strains containing chromosomal insertions of the transposon
Tn917 (34). The PBS-1 phage lysates were passed through the donor strains at
least three times before transduction of the recipient strain. Erythromycin and
lincomycin were used to select for resistance to macrolide, lincomycin, and
streptogramin B antibiotics (MLSr) encoded by the transposon.
MetK sequence comparisons. The tblastn program of the BLAST suite of
sequence analysis programs (1) was used to identify MetK homologs in unannotated sequence data. Signature amino acid patterns for SAM synthetases were
retrieved from the PROSITE database of protein families and domains (6).
␤-Galactosidase measurements. Strains containing lacZ fusions were grown in
Spizizen minimal medium containing all required amino acids at 50 ␮g/ml until
early exponential growth. The cells were harvested by centrifugation and then
resuspended in fresh Spizizen minimal medium in the presence or absence of
methionine. Samples were collected at 1-h intervals and assayed for ␤-galactosidase activity using toluene permeabilization (22). All starvation experiments
and assays were carried out at least twice, and variation was ⬍10%.
SAM synthetase assay. Cells (400 ml) were grown in Spizizen minimal medium
containing the required amino acids until the A600 reached 0.8. Cell extracts were
prepared by passage through a French pressure cell (8,000 lb/in2) followed by
removal of cell debris by centrifugation (22,000 ⫻ g for 20 min at 4°C). Protein
concentration of cell extracts was measured by the Bradford protein assay (BioRad) using a bovine serum albumin standard. Cell extracts were assayed for
SAM synthetase activity by measuring incorporation of [35S]methionine (1,175
Ci/mmol; Perkin-Elmer) into [35S]SAM using a protocol adapted from the work
of Ochi and Freese (24). Samples were filtered through P81 phosphocellulose
paper (Upstate Biotechnology) and washed with distilled water. Bound
[35S]SAM was quantitated in Packard Bioscience Ultima Gold scintillation fluid
using a Packard Tri-Carb 2100TR liquid scintillation counter. The recovery of
SAM after the wash with distilled water was 67% as determined with a [methyl14
C]SAM (ICN) standard; the measured amounts of [35S]SAM were therefore
multiplied by 1.5. The amount of [35S]SAM generated in the reaction mixture
was measured at 10-min intervals and used to determine the specific activity of
SAM synthetase in the crude cell extract by calculating the amount of [35S]SAM
(in pmol) generated per mg of protein in the cell extract per minute.
SAM-dependent transcription termination in vitro. DNA templates containing the B. subtilis yitJ leader region fused to the glyQS promoter were generated
by PCR, and single-round transcription reactions were carried out using Histagged B. subtilis RNAP as previously described (20, 21). Transcription reactions
were terminated by extraction with phenol, and products were resolved by denaturing polyacrylamide gel electrophoresis and visualized by PhosphorImager
(Molecular Dynamics) analysis. Percent termination was calculated from the
percentage of product in the terminated band relative to the total of the terminated and readthrough products. Reproducibility was ⫾5%. Extracts were prepared from cells grown to late logarithmic growth phase in minimal medium in
the presence of methionine. Cells were collected by filtration and extracted with
1.5 ml of 0.5 M formic acid, and the formic acid was removed by lyophilization
as described by Ochi et al. (25). Extracts were neutralized with KOH prior to
addition to the in vitro transcription termination assay. A standard curve for
SAM-dependent transcription termination was generated using diluted SAM
stocks neutralized with KOH.
RESULTS
Isolation of mutants exhibiting increased expression of Sbox genes during growth in the presence of methionine. The
observation that B. subtilis relA mutants exhibit partial methionine auxotrophy (37) and reduced S-box gene expression
(unpublished results) suggested that the stringent response
might influence S-box gene regulation. Since a possible requirement for the stringent response could prevent S-box gene
expression in the absence of amino acid starvation, we identified a suppressor mutant (sra-3, suppressor of RelA) in which
the methionine auxotrophy of a relA1 mutation was relieved
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FIG. 1. The S-box regulatory system. In the absence of SAM, the
antiterminator structure forms (AT, red-blue), preventing formation
of the competing terminator helix (T, blue) and allowing transcription
of the downstream coding sequence. Binding of SAM to the helix 1 to
4 region results in stabilization of helix 1, which serves as an antiantiterminator (AAT, black-red) by sequestering sequences necessary
for formation of the antiterminator, thereby allowing formation of the
terminator helix and transcription termination. SAM binding also promotes a tertiary interaction (dashed line) between residues (green) in
the terminal loop of helix 2 and the unpaired region between helices 3
and 4. SAM (*) is generated from methionine and ATP by MetK
(SAM synthetase).
3675
3676
MCDANIEL ET AL.
J. BACTERIOL.
TABLE 1. Specificity of the SBD1 allele
␤-Galactosidase activity for strain :
a
lacZ fusion construct
yitJ-lacZ
ykrT-lacZ
PtyrS-TrpsD-lacZ
tyrS-lacZ
yitJ-Pst3-lacZ
IS56B
IS56B-SBD1
0.10
0.14
5.3
32
0.11
60
14
7.4
19
0.13
TABLE 2. Effect of the SBD1 allele on expression of a yitJ-lacZ
transcriptional fusion and growth
Strain
BR151
BR151-SBD1
BR151MA
a
Fusions were integrated in single copy in strains IS56B and IS56B-SBD1
(metK10). Cells were grown in 2XYT medium until late logarithmic growth and
assayed for ␤-galactosidase activity (Miller units) (22).
Methionine
addition
yitJ-lacZ
expressiona
Doubling time
(min)
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫺
⫹
⫺
⫹
⫺
⫹
⫺
⫹
120
0.38
180
50
64
0.14
44
40
NDb
58
ND
60
84
76
130
134
a
Cells of strains BR151 and BR151-SBD1 (metB10) or BR151MA and
BR151MA-SBD1 (Met⫹) were grown in minimal medium containing methionine, harvested by centrifugation, and resuspended in minimal medium in the
presence (⫹) or absence (⫺) of methionine. yitJ-lacZ fusion activity is in Miller
units (22) and is reported for the time points at which maximal expression was
observed (4 h after the cultures were split for BR151 and BR151-SBD1 and 1 h
after the cultures were split for BR151MA and BR151MA-SBD1).
b
ND, not determined.
moved. Reintroduction of an unmutagenized yitJ-lacZ fusion
into the mutant strain background resulted in high expression
during growth in rich medium (Table 1), confirming that the
SBD1 mutation acts in trans to confer increased expression of
a wild-type fusion. Similarly, a second S-box gene fusion (ykrTlacZ) (9) exhibited a 100-fold increase in expression relative to
the wild-type parent, which indicates that the SBD1 mutation
is not specific for yitJ expression. In contrast, the SBD1 allele
had no effect on expression of a PtyrS-TrpsD-lacZ fusion containing a heterologous terminator (from the B. subtilis rpsD
gene, which encodes ribosomal protein S4) or a tyrS-lacZ fusion, which is regulated by the T-box transcription termination
control mechanism and responds to tRNATyr (8), demonstrating that the SBD1 allele does not generally increase
readthrough of leader region terminators.
The effect of the SBD1 allele on expression of a yitJ-lacZ
fusion containing a mutation in the antiterminator element was
tested to see whether the increased expression is dependent on
the antitermination control system. The yitJ-Pst3 mutation
(which alters residues in the 3⬘ side of helix 1, preventing
formation of the antiterminator and leaving the terminator
intact) results in loss of yitJ-lacZ expression under any growth
condition (9). The SBD1 allele did not increase expression of
a yitJ-lacZ fusion containing the yitJ-Pst3 allele, demonstrating
that the effect of the SBD1 allele is dependent on the normal
S-box termination control mechanism.
Effect of the SBD1 allele on growth and yitJ-lacZ expression.
The SBD1 allele was introduced into strains BR151 (metB10)
and BR151MA (Met⫹) to test the effect of the mutation on
growth and yitJ-lacZ expression levels in an unmutagenized
background and in the absence of the relA1 and sra-3 alleles. In
the Met⫺ strain BR151, expression of the yitJ-lacZ fusion was
high in cells starved for methionine for both the wild type and
the SBD1 mutant; the major difference between the strains was
that repression during growth in the presence of methionine
was reduced from ⬎300-fold for the wild-type strain to 3.5-fold
for the SBD1 mutant (Table 2). In the Met⫹ strain BR151MA,
removal of methionine from the growth medium resulted in a
transient induction of yitJ-lacZ expression (64 Miller units),
followed by adjustment to a steady-state level of expression of
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but S-box gene regulation was maintained. Four independent
pools of B. subtilis strain IS56B::yitJ-lacZ (lys-3 metB relA1
sra-3 strA SP␤::yitJ-lacZ) were randomly mutagenized with
EMS. The mutagenized cells were screened for isolates that
formed blue colonies on TBAB (a rich medium that contains
methionine) containing X-Gal, indicating loss of repression of
the yitJ-lacZ fusion during growth in the presence of methionine. Rich medium was chosen to allow growth of mutants that
might potentially affect multiple riboswitch systems and therefore might affect growth in minimal medium. At least one
mutant exhibiting high ␤-galactosidase activity was obtained
from each pool, with a total of 10 isolates from approximately
7,000 colonies.
Previous studies demonstrated that mutations in the helix 1
to 4 region or the terminator of the yitJ-lacZ fusion result in
loss of repression during growth in the presence of methionine
(9, 21, 38; unpublished results). To screen for isolates containing cis-acting mutations, chromosomal DNA was prepared for
each IS56B variant and introduced into a clean genetic background (B. subtilis strain ZB307Spc) by transformation, selecting for recombination of the SP␤ sequences flanking the yitJlacZ fusion in the chromosome of the mutant strains with the
resident SP␤ prophage in strain ZB307Spc. Eight of the 10
isolates were eliminated at this stage since introduction of the
yitJ-lacZ fusion from these strains into ZB307Spc produced
variants that formed blue colonies on TBAB containing X-Gal,
indicating that the mutation leading to expression during
growth in the presence of methionine was in the yitJ-lacZ
fusion.
The two remaining isolates were tested to determine
whether the ␤-galactosidase activity was derived from the yitJlacZ fusion rather than from expression of lacA, a cryptic
␤-galactosidase gene carried on the B. subtilis chromosome
(26). The yitJ-lacZ fusion in these isolates was replaced with a
neomycin resistance gene cassette by homologous recombination, and the resulting colonies were screened for lacZ expression. One isolate formed blue colonies on TBAB containing
X-Gal after removal of the yitJ-lacZ fusion, indicating that lacZ
expression in this variant was independent of the fusion and
probably resulted from expression of lacA. The mutation in the
remaining isolate was designated “S-box-derepressed mutation
1” (SBD1), as it resulted in derepression of S-box gene expression during growth in the presence of methionine.
Specificity of the SBD1 allele. The specificity of the effect of
the SBD1 allele on S-box gene expression was tested by introducing a series of transcriptional fusions into the chromosome
of IS56B-SBD1 from which the yitJ-lacZ fusion had been re-
BR151MA-SBD1
metB
VOL. 188, 2006
B. SUBTILIS SAM SYNTHETASE MUTANT
3677
approximately 15 to 20 Miller units, as previously reported (9,
20), while continuous growth in the presence of methionine
resulted in very low expression (⬍0.2 Miller units). In contrast,
introduction of the SBD1 allele resulted in constitutive high
expression (Table 2). These results indicate that the SBD1
allele is sufficient to confer loss of repression of S-box gene
expression during growth in the presence of methionine. The
SBD1 allele also resulted in a modest reduction in growth rate
in the BR151MA strain background, and this effect was not
suppressed by addition of methionine. Growth was not impaired in the BR151 background, which suggests that the
metB10 allele, which causes a defect in homoserine O-acetyltransferase and blocks the methionine biosynthesis pathway (10),
suppresses the growth defect caused by the SBD1 mutation.
Identification of the SBD1 allele. The SBD1 allele was genetically mapped using a set of B. subtilis mapping strains, each
of which has an insertion of Tn917 (conferring MLSr) at a
unique location in the chromosome (34). Generalized transduction using phage PBS-1 was employed to determine the
linkage between the SBD1 allele and the MLSr marker from
each mapping strain. PBS-1 transducing lysates were generated
in each of the mapping strains and used to transduce
BR151-SBD1::yitJ-lacZ. MLSr transductants were screened for
formation of white colonies on TBAB containing X-Gal, indicating replacement of the SBD1 allele in the recipient strain
with the wild-type allele by cotransduction with the MLSr
marker from the donor strain. All of the MLSr transductants
obtained formed blue colonies with the exception of transductants obtained with donor strain 1A642, in which the Tn917
insertion is 85% linked to ald (34), located at 3,277.3 kb on the
B. subtilis chromosome (16). Approximately 60% of the MLSr
transductants obtained using lysates from strain 1A642 formed
white colonies on TBAB containing X-Gal, indicating that the
mutation responsible for loss of repression during growth in
rich medium is located in the region of the chromosome near
the Tn917 insertion site of strain 1A642. Examination of the B.
subtilis genome sequence for candidate regulatory genes in this
region revealed that the metK gene, encoding SAM synthetase,
is located at 3,128.1 kb (16). Since mutations in metK (which is
itself an S-box gene) might be predicted to affect S-box gene
expression by affecting SAM pools, we tested whether the
SBD1 allele represents an alteration in the metK gene.
PCR products covering the entire metK gene, including its
promoter and leader region, were generated from chromosomal DNA from strains BR151 and BR151-SBD1, and the
DNA sequence was determined. The only change observed was
a single nucleotide substitution in the metK coding region that
corresponds to replacement of alanine with threonine at amino
acid position 83 (A83T). A 350-bp fragment of the metK coding
sequence containing the A83T substitution was isolated from
strain BR151-SBD1 by PCR and cloned into plasmid pGEM7Zf(⫹),
and the resulting construct was introduced into BR151::yitJ-lacZ
by transformation. Transformants were screened for formation of
blue colonies on TBAB containing X-Gal, indicating introduction
of the SBD1 allele by homologous recombination. Chromosomal
DNA was isolated from transformants that exhibited either high
or low yitJ-lacZ expression, and the metK locus was isolated by
PCR and screened for the presence of the A83T substitution by
DNA sequencing. All transformants that formed white colonies
on TBAB containing X-Gal were wild type for metK, while all
transformants that formed blue colonies (indicating loss of repression during growth in the presence of methionine) contained
the A83T substitution. These results demonstrated that the A83T
substitution in metK is sufficient to confer the phenotype observed
for the SBD1 allele. We therefore designate this mutation
metK10.
MetK homologs were identified by tblastn searches (1) in 69
additional bacterial species representing 46 different genera.
The sequences present in the region surrounding A83 from B.
subtilis MetK and the MetKs from other representative species
are shown in Fig. 2. The identity of the amino acid analogous
to A83 in B. subtilis MetK was alanine in 28 species representing 16 genera, serine in 32 species representing 25 genera,
threonine in 8 species representing 4 genera, and glycine in one
Downloaded from http://jb.asm.org/ on February 28, 2014 by PENN STATE UNIV
FIG. 2. Sequence comparison of bacterial MetK homologs. The region of B. subtilis MetK surrounding residue A83 is aligned with the
analogous MetK regions from other bacterial species. Sequences were obtained by searching the NCBI database with signature sequences for SAM
synthetases (6) by using the tblastn program (1). A83 in B. subtilis MetK and the analogous residues from the other bacterial MetKs are boxed.
Residues that differ from the B. subtilis sequence are shown in boldface. Bsu, B. subtilis; Ban, Bacillus anthracis; Sau, Staphylococcus aureus; Lpl,
Lactobacillus plantarum; Spn, Streptococcus pneumoniae; Sty, Salmonella enterica serovar Typhimurium; Bma, Burkholderia mallei; Pae, Pseudomonas aeruginosa; Bpe, Bordetella pertussis; Eco, E. coli. (⫹), gram-positive species; (⫺), gram-negative species. Amino acid identities to B. subtilis
MetK were as follows: 86%, B. anthracis; 77%, S. aureus; 68%, L. plantarum; 68%, S. pneumoniae; 63%, S. enterica serovar Typhimurium; 59%,
B. mallei; 59%, P. aeruginosa; 58%, B. pertussis; 57%, E. coli.
3678
MCDANIEL ET AL.
organism. Of the 28 species containing alanine at this position
in MetK, 20 are gram positive, including six additional Bacillus
species. All MetK variants containing serine at this position are
from gram-negative organisms. Of the eight species containing
threonine at this position, six species are gram negative; the
two gram-positive species are Streptococcus mutans and Streptococcus pneumoniae, both of which use a SAM-binding riboswitch (the SMK box) distinct from the S box to regulate metK
expression in response to SAM (5). The A83T mutation in the
metK10 allele appears to be a conservative change, as threonine is found at the corresponding position in other metK
genes.
Measurement of SAM synthetase activity in BR151-SBD1.
The identification of the SBD1 mutation as a single amino acid
substitution in metK suggested that the reduced repression of
S-box gene expression could be due to a reduction in SAM
synthetase activity. The level of SAM synthetase activity in
strains BR151 and BR151-SBD1 grown in minimal medium
containing methionine was therefore measured. SAM synthetase activity in strain BR151 was 150 ⫾ 7 pmol min⫺1 mg of
protein⫺1, consistent with the range of activity (128 to 200
pmol min⫺1 mg of protein⫺1) previously reported for metK⫹
strains of B. subtilis (24, 35, 40). Activity in strain BR151-SBD1
was reduced 15-fold to 10 ⫾ 2 pmol min⫺1 mg of protein⫺1. As
noted above, the SBD1 allele had no effect on growth rate in
the BR151 strain background (Table 2). Other metK mutant
strains with a similar reduction of SAM synthetase activity also
exhibited normal growth in medium containing methionine
(35), suggesting that the amount of SAM produced under
these conditions is sufficient for growth but not for repression
of S-box gene expression.
Effect of the metK10 allele on SAM pools in vivo. Transcription termination at the B. subtilis yitJ leader region terminator
occurs in response to the addition of SAM to an in vitro
transcription assay (20, 21). We used this assay to monitor the
SAM concentration in cell extracts of wild-type and metK10
mutant cells grown in the presence of methionine by comparing the termination efficiency promoted by addition of these
cell extracts to the transcription termination assay to the termination efficiency promoted by known concentrations of
SAM. As reported previously (21), addition of 0.15 ␮M SAM
resulted in 50% termination (data not shown). The wild-type
cell extract was fourfold more efficient in promotion of transcription termination than was the mutant cell extract (Fig. 3),
corresponding to SAM pools of 250 ␮M for the wild-type strain
versus 59 ␮M for the metK10 mutant. The SAM concentration
(measured by high-pressure liquid chromatography) for metK⫹
strains grown in the presence of methionine was previously
reported at 400 ␮M (35), a value similar to that obtained using
the in vitro transcription termination assay. This assay therefore provides a convenient method for monitoring SAM concentrations in crude cell extracts.
DISCUSSION
SAM has been shown to cause transcription termination of
S-box genes in vitro, and overexpression of SAM synthetase
results in decreased S-box gene expression in vivo (20). In this
study we attempted to isolate trans-acting mutations that lead
to loss of repression of S-box gene expression during growth in
the presence of methionine and predicted that these mutations
could occur in genes that affect SAM pools or in genes encoding other unknown factors that are involved in S-box gene
regulation in vivo. A single trans-acting mutation (SBD1 or
metK10) was isolated that led to derepression of S-box gene
expression, and this mutation was identified as a single nucleotide substitution in the metK gene, which encodes SAM synthetase. The observation that this mutation results in reduced
SAM synthetase activity and SAM pools during growth in the
presence of methionine further supports the model that the
S-box RNAs directly monitor SAM in vivo. No evidence was
obtained for participation of additional cellular factors in the
S-box mechanism, but it is possible that mutations of other
types were missed.
The metK gene product converts methionine and ATP into
SAM, which is required for methyltransferase reactions in the
cell as well as for polyamine biosynthesis (19). The availability
of methionine in the cell can therefore be measured indirectly
by sensing SAM levels, as methionine must be present for
SAM to be synthesized. SAM is the key effector controlling
methionine biosynthesis gene expression in Escherichia coli at
the level of transcription initiation (7), indicating that SAM is
the effector of choice in a variety of biological systems despite
variability in the regulatory mechanism. The metK10 allele
results in a 15-fold reduction in SAM synthetase activity and a
4-fold reduction in SAM pools. Mutations with reduced SAM
Downloaded from http://jb.asm.org/ on February 28, 2014 by PENN STATE UNIV
FIG. 3. Measurement of SAM pools in cell extracts of metK⫹ and
metK10 strains by SAM-dependent transcription termination. Cell extracts were generated from strains BR151MA and BR151MA-SBD1
by formic acid extraction of cells grown in minimal medium in the
presence of methionine. Extracts were neutralized by the addition of 1
N KOH, and samples (standardized by cell numbers used in preparation of the extracts) were added to a B. subtilis RNAP in vitro transcription reaction mixture containing a Pgly-yitJ DNA template. The
percent termination was calculated as the fraction of the terminated
RNA product relative to the total of the terminated and readthrough
products. Reproducibility was ⫾5%. Measurement of termination efficiency in response to addition of various amounts of SAM resulted in
generation of a standard curve in which 50% termination was observed
at 0.15 ␮M SAM. The amount of cell extract required to confer 50%
termination was compared to the SAM standard curve. An average
internal cell volume of 0.535 ⫾ 0.13 ␮l A600⫺1 (35) was used to calculate the intracellular SAM concentrations (250 ␮M for BR151MA and
59 ␮M for BR151MA-SBD1) from the cell equivalents and A600 of cell
cultures used in preparation of the extracts.
J. BACTERIOL.
VOL. 188, 2006
B. SUBTILIS SAM SYNTHETASE MUTANT
3679
synthetase activity were previously shown to result in an increase in intracellular methionine, while overexpression of
SAM synthetase results in partial methionine auxotrophy (40).
These results are consistent with the prediction that SAM is
the molecular effector for S-box gene expression both in vivo
and in vitro (3, 20).
Inactivation of the metK gene is a lethal event in B. subtilis,
as some level of SAM synthetase activity is essential for viability (40). It was therefore possible only to isolate mutations
that decrease SAM synthetase activity, not abolish it. Wabiko
et al. (35) identified metK mutant strains of B. subtilis that
resulted in a 25- to 200-fold decrease in SAM synthetase activity compared to the parent strain, yet these strains exhibited
only a three- to fourfold decrease in SAM pools during growth
in the presence of methionine, consistent with the results obtained for the metK10 allele. It therefore appears that a significant reduction in SAM synthetase activity can be tolerated, as
only the most severe mutations result in a growth defect. A
major decrease in SAM pools has a variety of effects in E. coli,
including poor growth and increased mutation rate, due at
least in part to defects in DNA methylation (27, 36). The
metK10 mutation resulted in a reduction in SAM pools and
growth rate that was not suppressed by supplementation of the
growth medium with extra methionine. The observation that
the growth defect occurred only in a metB⫹ strain and not in a
metB10 mutant suggests that this effect is not due to limitation
for SAM but instead results from a metabolic imbalance that
occurs as a consequence of deregulation of the methionine
biosynthesis pathway by disruption of SAM-dependent repression of multiple S-box genes; this imbalance, which could result
in both accumulation of toxic intermediates and draining of
key precursors, is absent in a metB mutant that lacks homoserine O-acetyltransferase and is therefore blocked at the first
step of the pathway (10). The B. subtilis metK gene is also
predicted to be a member of the S-box regulon (9), suggesting
that metK expression is likely to increase when SAM pools are
low. B. subtilis metK expression increases during methionine
limitation (3, 40), and metK genes from members of the Lactobacillales are regulated by SAM via the SMK box, a SAMresponsive riboswitch distinct from the S box (5). Increased
expression of metK in metK10 strains may partially compensate
for the reduction in SAM synthetase activity caused by the
mutation.
The amino acid sequence of SAM synthetase enzymes is
highly conserved, and B. subtilis and E. coli SAM synthetases
exhibit 57% amino acid identity. E. coli SAM synthetase has
been characterized in detail and consists of four identical subunits (17). Two subunits form a tight dimer with two active
sites located between them, and two dimers interact in an
asymmetrical arrangement to form a peanut-shaped tetrameric
enzyme (32). The interactions between the dimers appear to be
less extensive than the subunit-subunit interactions within each
Downloaded from http://jb.asm.org/ on February 28, 2014 by PENN STATE UNIV
FIG. 4. Location of S80 in the crystal structure of the E. coli SAM synthetase ternary complex. The crystal structure of E. coli SAM synthetase
(15) with the ATP analog AMPPNP and methionine in each active site is shown. The image was created using the Cn3D v4.1 three-dimensional
structure viewer (NCBI), using coordinates from the work of Komoto et al. (15). The tetrameric enzyme is a dimer of dimers, with two active sites
located between the two subunits in each dimer. The location of S80 (in tan) in each subunit is indicated by red arrows. S80 is the only residue
that participates in hydrogen bond formation between the dimers of E. coli SAM synthetase.
3680
MCDANIEL ET AL.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
ACKNOWLEDGMENTS
26.
This work was supported by National Institutes of Health grant
GM63615 and by National Institutes of Health predoctoral fellowship
F32 GM20923 (to B.A.M.).
REFERENCES
1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.
Basic alignment search tool. J. Mol. Biol. 215:403–410.
2. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741–746.
3. Auger, S., A. Danchin, and I. Martin-Verstraete. 2002. Global expression
profile of Bacillus subtilis grown in the presence of sulfate or methionine. J.
Bacteriol. 184:5179–5186.
4. Epshtein, V., A. S. Mironov, and E. Nudler. 2003. The riboswitch-mediated
control of sulfur metabolism in bacteria. Proc. Natl. Acad. Sci. USA 100:
5052–5056.
5. Fuchs, R. T., F. J. Grundy, and T. M. Henkin. 2006. The SMK box is a new
SAM-binding RNA for translational regulation of SAM synthetase. Nat.
Struct. Mol. Biol. 13:226–233.
6. Gasteiger, E., A. Gattiker, C. Hoogland, I. Ivanyi, R. D. Appel, and A.
Bairoch. 2003. ExPasy: the proteomics server for in-depth protein knowledge
and analysis. Nucleic Acids Res. 31:3784–3788.
7. Greene, R. C. 1996. Biosynthesis of methionine, p. 542–560. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik,
W. S. Reznikoff, M. Riley, M. Schaecter, and H. E. Umbarger (ed.), Esch-
27.
28.
29.
30.
31.
32.
33.
34.
35.
erichia coli and Salmonella: cellular and molecular biology, 2nd ed. American
Society for Microbiology, Washington, D.C.
Grundy, F. J., and T. M. Henkin. 1993. tRNA as a positive regulator of
transcription antitermination in B. subtilis. Cell 74:475–482.
Grundy, F. J., and T. M. Henkin. 1998. The S box regulon: a new global
transcription termination control system for methionine and cysteine biosynthesis genes in Gram-positive bacteria. Mol. Microbiol. 30:737–749.
Grundy, F. J., and T. M. Henkin. 2002. Synthesis of serine, glycine, cysteine,
and methionine, p. 245–254. In A. L. Sonenshein, J. A. Hoch, and R. Losick
(ed.), Bacillus subtilis and its closest relatives: from genes to cells. American
Society for Microbiology, Washington, D.C.
Grundy, F. J., and T. M. Henkin. 2003. The T box and S box transcription
termination control systems. Front. Biosci. 8:d20–d31.
Grundy, F. J., and T. M. Henkin. 2004. Regulation of gene expression by
effectors that bind to RNA. Curr. Opin. Microbiol. 7:126–131.
Grundy, F. J., D. A. Waters, S. H. Allen, and T. M. Henkin. 1993. Regulation
of the Bacillus subtilis acetate kinase gene by CcpA. J. Bacteriol. 175:348–
355.
Henkin, T. M., and G. H. Chambliss. 1984. Genetic mapping of a mutation
causing an alteration in Bacillus subtilis ribosomal protein S4. Mol. Gen.
Genet. 193:364–369.
Komoto, J., T. Yamada, Y. Takata, G. D. Markham, and F. Takusagawa.
2004. Crystal structure of the S-adenosylmethionine synthase ternary complex: a novel catalytic mechanism of S-adenosylmethionine synthesis from
ATP and Met. Biochemistry 43:1821–1831.
Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, et al. 1997.
The complete genome sequence of the Gram-positive bacterium Bacillus
subtilis. Nature 390:249–256.
Markham, G. D., E. W. Hafner, C. W. Tabor, and H. Tabor. 1980. SAdenosylmethionine synthetase from Escherichia coli. J. Biol. Chem. 255:
9082–9092.
Markham, G. D., and C. Satishchandran. 1988. Identification of the reactive
sulfhydryl groups of S-adenosylmethionine synthetase. J. Biol. Chem. 263:
8666–8670.
Matthews, R. G. 1996. One-carbon metabolism, p. 600–611. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik,
W. S. Reznikoff, M. Riley, M. Schaecter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American
Society for Microbiology, Washington, D.C.
McDaniel, B. A. M., F. J. Grundy, I. Artsimovitch, and T. M. Henkin. 2003.
Transcription termination control of the S box system: direct measurement
of S-adenosylmethionine by the leader RNA. Proc. Natl. Acad. Sci. USA
100:3083–3088.
McDaniel, B. A., F. J. Grundy, and T. M. Henkin. 2005. A tertiary structural
element in S box leader RNAs is required for S-adenosylmethionine-directed transcription termination. Mol. Microbiol. 57:1008–1021.
Miller, J. 1972. Experiments in molecular genetics. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.
Nudler, E., and A. S. Mironov. 2004. The riboswitch control of bacterial
metabolism. Trends Biochem. Sci. 29:11–17.
Ochi, K., and E. Freese. 1982. A decrease in S-adenosylmethionine synthetase activity increases the probability of spontaneous sporulation. J. Bacteriol. 152:400–410.
Ochi, K., J. C. Kandala, and E. Freese. 1981. Initiation of Bacillus subtilis
sporulation by the stringent response to partial amino acid deprivation.
J. Biol. Chem. 256:6866–6875.
Ogura, M., S. Hirao, Y. Ohshiro, and T. Tanaka. 1999. Positive regulation of
Bacillus subtilis sigD by C-terminal truncated LacR at translational level.
FEBS Lett. 457:112–116.
Posnick, L. M., and L. D. Samson. 1999. Influence of S-adenosylmethionine
pool size on spontaneous mutation, Dam methylation, and cell growth of
Escherichia coli. J. Bacteriol. 181:6756–6762.
Reczkowski, R. S., and G. D. Markham. 1995. Structural and functional roles
of cysteine 90 and cysteine 240 in S-adenosylmethionine synthetase. J. Biol.
Chem. 270:18484–18490.
Rodionov, D. A., A. G. Vitreschak, A. A. Mironov, and M. S. Gelfand. 2004.
Comparative genomics of the methionine metabolism in Gram-positive bacteria: a variety of regulatory systems. Nucleic Acids Res. 32:3340–3353.
Smith, I., P. Paress, K. Cabane, and E. Dubnau. 1980. Genetics and physiology of the rel system of Bacillus subtilis. Mol. Gen. Genet. 178:272–279.
Steinmetz, M., and R. Richter. 1994. Plasmids designed to alter the antibiotic
resistance expressed by insertion mutations in Bacillus subtilis. Gene 142:
79–83.
Takusagawa, F., S. Kamitori, S. Misaki, and G. D. Markham. 1996. Crystal
structure of S-adenosylmethionine synthetase. J. Biol. Chem. 271:136–147.
Tucker, B. J., and R. R. Breaker. 2005. Riboswitches as versatile control
elements. Curr. Opin. Struct. Biol. 15:342–348.
Vandeyar, M. A., and S. A. Zahler. 1986. Chromosomal insertions of Tn917
in Bacillus subtilis. J. Bacteriol. 167:530–534.
Wabiko, H., K. Ochi, D. M. Nguyen, E. R. Allen, and E. Freese. 1988. Genetic
mapping and physiological consequences of metE mutations of Bacillus subtilis. J. Bacteriol. 170:2705–2710.
Downloaded from http://jb.asm.org/ on February 28, 2014 by PENN STATE UNIV
dimer (15, 32). Residue S80 in E. coli MetK, which corresponds to the position of the A83T substitution of the B.
subtilis metK10 allele, is located at the tetramer interface and is
the only residue that participates in hydrogen bond formation
between the dimers (32) (Fig. 4). Substitution of the nonpolar
alanine residue with a polar threonine residue at position 83 in
B. subtilis MetK could affect activity by affecting tetramerization, which is required for full activity (18). The presence of
threonine at this position in MetK proteins from certain species suggests that sequence context may influence its role.
Modification of both C90, a highly conserved residue located at
the tetramer interface, and C240 by N-ethylmaleimide results
in dissociation of E. coli SAM synthetase active tetramers into
inactive dimers. Site-directed mutagenesis of C90 to alanine or
serine results in a mixture of dimers and tetramers and a
reduction in SAM synthetase activity; the purified dimers exhibit approximately 20-fold-lower activity than the purified tetramers (28), a reduction in activity similar to that observed for
the metK10 allele. Further characterization will be necessary to
determine if the reduction in SAM synthetase activity conferred by the metK10 allele is due to an effect on oligomerization, on some other functional property of the enzyme, or on
protein stability.
The metK10 mutation was the only trans-acting mutation
identified in this study. While it is possible that SAM, which is
sufficient to cause transcription termination in vitro, may be
the only factor involved in S-box gene regulation in vivo, it
remains possible that other cellular components are involved.
We predicted that mutations resulting in a specific defect in
S-box gene expression would not be lethal, as the resulting
overexpression of genes involved in methionine biosynthesis
should be tolerated, especially in a metB10 genetic background
in which the methionine biosynthesis pathway is blocked; this
prediction is supported by the modest growth defect caused by
the metK10 allele even in a metB⫹ background, despite significant derepression of S-box gene expression. However, the
possibility remains that additional required gene products
could not be identified because they play essential roles in
other cellular processes.
J. BACTERIOL.
VOL. 188, 2006
36. Wei, Y., and E. B. Newman. 2002. Studies on the role of the metK gene
product of Escherichia coli K-12. Mol. Microbiol. 43:1651–1656.
37. Wendrich, T. M., and M. A. Marahiel. 1997. Cloning and characterization of
a relA/spoT homologue from Bacillus subtilis. Mol. Microbiol. 26:65–79.
38. Winkler, W. C., F. J. Grundy, B. A. Murphy, and T. M. Henkin. 2001. The
GA motif: an RNA element common to bacterial antitermination systems,
rRNA, and eukaryotic RNAs. RNA 7:1165–1172.
39. Winkler, W. C., A. Nahvi, N. Sudarsen, J. E. Barrick, and R. R. Breaker.
B. SUBTILIS SAM SYNTHETASE MUTANT
3681
2003. An mRNA structure that controls gene expression by binding Sadenosylmethionine. Nat. Struct. Biol. 10:701–707.
40. Yocum, R. R., J. B. Perkins, C. L. Howitt, and J. Pero. 1996. Cloning and
characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis. J. Bacteriol. 178:4604–4610.
41. Zuber, P., and R. Losick. 1987. Role of AbrB in Spo0A- and Spo0B-dependent utilization of a sporulation promoter in Bacillus subtilis. J. Bacteriol.
169:2223–2230.
Downloaded from http://jb.asm.org/ on February 28, 2014 by PENN STATE UNIV