The polymers of fructose, levan and inulin, as well as sucrose and
raffinose, are substrates for the product of the fruA gene of Streptococcus mutans GS-5. The purpose of this study was
to characterize the DNA immediately flanking fruA, to
explore the regulation of expression of fruA by the
carbohydrate source, and to begin to elucidate the molecular basis for
differential expression of the gene. Located 3' to fruA was
an open reading frame (ORF) with similarity to 
-fructosidases which
was cotranscribed with fruA. A transcriptional initiation
site, located an appropriate distance from an extended 
10-like
promoter, was mapped at 165 bp 5' to the fruA structural
gene. By the use of computer algorithms, two overlapping, stable
stem-loop sequences with the potential to function as rho-independent
terminators were found in the 5' untranslated region. Catabolite
response elements (CREs), which have been shown to govern carbon
catabolite repression (CCR) by functioning as negative cis
elements in gram-positive bacteria, were located close to the promoter.
The levels of production of fruA mRNA and FruA were
elevated in cells growing on levan, inulin, or sucrose as the sole
carbohydrate source, and repression was observed when cells were grown
on readily metabolizable hexoses. Deletion derivatives containing
fusions of fruA promoter regions, lacking sequences 5' or
3' to the promoter, and a promoterless chloramphenicol
acetyltransferase gene were used (i) to demonstrate the functionality
of the promoter mapped by primer extension, (ii) to demonstrate that
CCR of the fru operon requires the CRE that is located 3'
to the promoter region, and (iii) to provide preliminary evidence that
supports the involvement of an antitermination mechanism in
fruA induction.
 |
INTRODUCTION |
A variety of oral bacteria can
utilize sucrose to produce extracellular polysaccharides composed of
2,1- and 2,6-linked fructosyl moieties. For example,
Streptococcus salivarius and Actinomyces
naeslundii produce fructosyltransferases (FTFs) that synthesize
levan-type polymers, which are rich in 2,6 linkages, whereas the
FTFs of Streptococcus mutans and Streptococcus
sanguis produce inulin-type polymers, composed predominantly of
2,1-linked fructose (4). The ability to synthesize
fructan polymers within oral biofilms is believed to allow the
organisms to capture a greater proportion of dietary sucrose by
converting it to a nondiffusing, extracellular storage polysaccharide
which can be catabolized when exogenous sources are lacking (7,
21, 25). Consistent with this model is the observation that
organisms which produce fructans usually possess enzymes that are
capable of degrading fructan polymers.
S. mutans produces a secreted fructan hydrolase which is
encoded by the fruA gene (13). FruA releases
fructose from levan, inulin, and raffinose, and it cleaves sucrose into
glucose and fructose (13). Analysis of the deduced amino
acid sequence of FruA revealed that it is synthesized as a 158-kDa
protein, containing a signal sequence typical of gram-positive
bacteria, which is presumably cleaved to yield a 155-kDa mature,
secreted enzyme (9). The central one-third of the FruA
enzyme exhibits similarity to fructan-hydrolyzing enzymes from
eubacteria and lower eukaryotes. The N and C termini exhibit no
significant similarity to other known proteins, with one exception: an
LPXTG anchoring sequence is found at the C terminus, consistent with
the finding that the enzyme can be found in significant quantities in
association with the cell surface under certain environmental
conditions (11). Isogenic mutants were used in a program-fed
rat caries model to show that FruA is a virulence determinant that
contributes to the progression of dental caries (8).
Results from early studies on the control of fructan hydrolase
expression by S. mutans indicated that the activity of this enzyme was increased in supernatant fluids of cell cultures grown on
fructans and sucrose (30, 55) compared with that of cells grown on glucose. Cultivation of S. mutans with combinations
of both inducing substrates and readily metabolizable hexoses yielded levels of activity similar to those obtained with hexose alone. Also,
fructan hydrolase activity was found to be higher when fructose was
provided as the limiting carbohydrate in continuous culture than when
glucose was the growth carbohydrate (30). More recently, it
has been found that although many strains of S. mutans
produce only FruA, some strains of S. mutans can produce two
fructan-hydrolyzing activities. The major one is FruA, and the second
activity is an inulinase with no detectable activity on levans
(33). The environmental factors specifically regulating
fruA expression and the molecular basis for the apparent
differential expression of fructan hydrolase activity remain to be
elucidated. In a preliminary report, we noted that fruA
expression appeared to be inducible by inulin and was repressible by
readily metabolizable hexoses (10). This communication
presents the results of a study designed to examine the transcriptional
organization of the fruA operon, which includes a second
gene with similarity to fructosidase genes, and to uncover the basic
molecular mechanisms for control of induction and repression of
fruA.
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MATERIALS AND METHODS |
Bacterial strains and media.
S. mutans strains were
maintained on brain heart infusion (BHI) agar (Difco) containing
antibiotics when indicated. Genetically competent S. mutans
was prepared as previously described (43), and transformants
were selected on BHI agar supplemented with tetracycline (5 µg/ml),
erythromycin (10 µg/ml), or spectinomycin (250 µg/ml). For
induction and repression experiments, and for studies involving
chloramphenicol acetyltransferase (CAT) reporter gene fusion
measurements, S. mutans was grown to an optical density at
600 nm (OD600) of ca. 0.5 to 0.7 in a tryptone-vitamin base (TV) medium 3.5% tryptone with 0.04 µg of p-aminobenzoic
acid/ml, 0.2 µg of thiamine-HCl/ml, 1 µg of nicotinamide/ml, and
0.2 µg of riboflavin/ml) which was supplemented with 0.5% (wt/vol)
levan (prepared as detailed below), inulin (from Dahlia
tubers; Sigma) or simple sugars, as well appropriate antibiotics when
necessary. TV medium was developed because tryptone-yeast extract base
medium (58) contained significant amounts of carbohydrate
and could support the growth of S. mutans to an
OD600 of ca. 0.3 to 0.4. S. mutans was not able
to grow in TV medium without carbohydrate supplementation, so
experiments designed to look at induction and repression of
fruA were not confounded by the presence of significant
quantities of uncharacterized sugars. Escherichia coli DH10B
was maintained on L agar. E. coli transformants were selected on L medium containing either tetracycline (5 µg/ml), ampicillin (100 µg/ml), spectinomycin (100 µg/ml), chloramphenicol (50 µg/ml), erythromycin (300 µg/ml), or combinations of
antibiotics when necessary. Medium components, excluding sugars, were
obtained from Difco, and chemicals were purchased from Sigma Chemical
Co. (St. Louis, Mo.). Levan was prepared from supernatant fluid of S. salivarius 57.1 cultures (52). The supernate
was collected and buffered at pH 6.5 with 10 mM potassium phosphate,
and phenylmethylsulfonyl fluoride and sodium azide were added to final
concentrations of 1 mM and 0.02%, respectively. Supernatant fluid was
concentrated approximately 20-fold at 4°C, using a membrane with a
molecular weight cutoff of 30,000. The concentrated material was placed in a dialysis bag with a 6,000-Mr cutoff and
dialyzed against 2 liters of 10 mM potassium phosphate, pH 6.5, containing 50 mM raffinose and 0.02% sodium azide for 2 to 3 days at
37°C. Raffinose is a substrate for FTF but not for the
glucosyltransferases produced by oral streptococci. Levan that
accumulated in the dialysis bag was ethanol precipitated, dissolved in
deionized H2O, reprecipitated with ethanol, and lyophilized
to dryness. To assess the purity of the levan, the polysaccharide was
hydrolyzed in 0.5 M acetic acid for 1 h at 70°C. The ketohexose
content was determined by the cysteine-H2SO4
method (20), total reducing sugar was measured by the
dinitrosalicylic acid assay (38) with fructose as a
standard, and total protein was determined by the method of Bradford
(6), using the Bio-Rad protein assay reagent. Levan thus
prepared was found to be >99% fructose with <0.5% protein contamination.
DNA manipulations.
Chromosomal DNA was prepared from
S. mutans as previously described (13).
Small-scale plasmid DNA isolations from S. mutans were
performed according to the method of Anderson and McKay
(1a), except that mutanolysin was added to a final
concentration of 20 U/ml during the lysozyme digestion step. For rapid
screening of recombinants, plasmid DNA from E. coli was
prepared by an alkaline lysis procedure (5). For
transformation of S. mutans, plasmid DNA was prepared from
E. coli by a rapid boiling method (39) to
minimize nicking of the DNA. For nucleotide sequencing, plasmid DNA was
prepared by alkaline lysis followed by polyethylene glycol purification
(39).
DNA sequencing was performed by the dideoxynucleotide chain termination
method (48). Primers used in sequencing were either M13
forward or reverse primers (U.S. Biochemicals) or custom primers based
on derived sequence data. Custom primers either were synthesized with
an Applied Biosystems model 391 DNA synthesizer or were purchased from
Life Technologies Inc. (LTI; Bethesda, Md.). Sequencing reactions were
performed by using Sequenase version 2.0 (U.S. Biochemicals) or a
Ladderman sequencing kit (Pan Vera, Madison, Wis.) with Bca thermostable DNA polymerase, as recommended by the manufacturer. Products of sequencing reactions were labeled with
[
-35S]dATP, separated by 6% denaturing polyacrylamide
gel electrophoresis, and subjected to autoradiography. Sequences were
analyzed with MacVector version 4.0.1 and AssemblyLIGN version 1.0.5 from IBI (New Haven, Conn.), with the University of Wisconsin Genetics Computer Group (GCG) Wisconsin Package version 8.1 DNA analysis software and a Vax workstation, and with BLAST search algorithms available through the National Center for Biotechnology Information (Bethesda, Md.).
Constructs containing DNA fragments from the 5' region of
fruA (see Fig. 6) were obtained by the synthesis and use of
primers designed to amplify the desired region from plasmid pFRU1
(13). The primers contained mismatched bases such that
BamHI and/or PstI sites were generated for
subsequent cloning of the PCR products onto pU1, which is pUC18
carrying an E. coli CAT gene (cat) (Pharmacia Biotech, Piscataway, N.J.) in the orientation opposite that of the
lac promoter. Positive recombinants were selected for their ability to confer chloramphenicol resistance to E. coli.
Sequencing reactions were performed to confirm orientation and
error-free PCR amplification. The construct, WHFRU, containing
sequences from nucleotides (nt) 564 to +101 with respect to the
translation initiation site (see Fig. 6), was cloned into the
streptococcal suicide vector pSU20Erm (31). The construct
was introduced into S. mutans, and transformants were
selected on medium containing erythromycin. Southern hybridization was
used to confirm integration of WHFRU at the fruA locus by
Campbell insertion. The 5' deletion constructs were transferred to the
E. coli-Streptococcus shuttle vector pDL278 (36),
and Spr transformants of S. mutans US100, a
recA derivative of S. mutans GS-5 constructed for
this study by insertional inactivation of recA with a
previously described construct (45), were selected.
Strain FCAT (see Fig. 6) was constructed by synthesizing primers at
positions 254 to 234 and 18 to +10 relative to the fruA translational start site. The primers contained
internal mismatched bases such that BamHI sites were
generated in the resulting PCR product at the 5' end and at position
1 relative to the translational start site of fruA.
Products, purified as described above, were ligated into pCW24
(16), a pUC-based vector containing a promoterless cat gene. A BamHI site was incorporated at the 5'
end of cat so that PCR products derived from the 5' end of
fruA and containing the cognate ribosome binding site (RBS)
of fruA could be correctly spaced from the start codon of
the cat gene (see Fig. 6). Positive recombinants were
selected for their ability to confer chloramphenicol resistance to
E. coli. Sequencing reactions were performed to confirm
orientation and sequence identity. The gene fusion cassette was
transferred to the suicide vector pSF143 (53). One positive clone, FCAT, was used to transform S. mutans, and Southern
hybridizations were performed to confirm integration at the
fruA locus by single-crossover insertion.
A 3' deletion construct was prepared by synthesizing an antisense
primer encompassing nt 254 to 156 (
CRE) relative to the fruA translational start site; this antisense primer,
containing a 5' overhang with a PstI site, was used in
conjunction with the sense primer employed in the construction of FCAT
to amplify products by PCR. Products were cloned directly into plasmid
pCR2.1 (Invitrogen), sequenced to confirm their identity, and then
cloned into pDLCAT, which is pDL278 in which the cat gene
from pU1 has been cloned in the orientation opposite that of the
lacZ promoter on pDL278, to create pDLCRE. Recombinants
were selected for their ability to confer both Spr and
Cmr to E. coli and used to transform S. mutans US100. Recombinant S. mutans strains were
screened by restriction analysis of small-scale preparations of
plasmids to ensure plasmid stability.
RNA manipulations.
For preparation of RNA, S. mutans was grown to mid-exponential phase in TV medium
supplemented with the desired carbohydrate at 0.5%. Total RNA from
S. mutans GS-5 was isolated as previously described
(15), using a modification of the protocol of Putzer et al.
(44). RNA samples were denatured and transferred to
nitrocellulose membranes (Schleicher and Schuell) by using a slot blot
apparatus (LTI), and the RNAs were UV cross-linked to the membranes and air dried. Blots were probed with DNA fragments that had been purified
after excision from agarose gels (2). The probes consisted of an 834-bp HincII fragment encompassing nt +724 to +1557
of the fruA structural gene (9) or a 1.12-kbp
internal NdeI fragment from fruB. Hybridizations
and washes were carried out under high-stringency conditions. Signals
on autoradiographs were quantified by using an IS1000 digital imaging
system from Alpha Innotech Corp. (San Leandro, Calif.).
The transcriptional start site of fruA was mapped by primer
extensions with four different primers, and the same result was achieved regardless of the primer used. Primers were end labeled, and
primer extension reactions were performed essentially as described previously (2, 31). DNA sequencing reactions were performed with the same primers and pFRU1 (16). Reverse transcriptase (RT) PCR was performed as described previously (16).
Enzyme preparation and assays.
Protein preparations from
exponentially growing cultures of S. mutans were assayed for
fructan hydrolase activity as previously described (11, 59).
One unit of activity was defined as the amount of enzyme needed to
release 1 µmol of reducing sugar per h. To prepare samples for CAT
assays, cleared lysates were prepared as previously detailed
(16) and used directly for determination of CAT activity
according to the protocol of Shaw (50). One unit of CAT
activity was defined as the amount of enzyme necessary to acetylate 1 nmol of chloramphenicol per min. The values expressed for all enzyme
assays are averages of data for three to six independently grown cell
cultures, and all assays were performed at least in triplicate.
Nucleotide sequence accession number.
The complete
nucleotide sequence of the fruAB genes and flanking regions
has been deposited with GenBank and bears accession no. AF093758.
| |
RESULTS |
Genetic organization of the fru operon.
The
complete nucleotide sequence of the fruA gene of S. mutans GS-5, including 685 nt 5' of the translational initiation
site and approximately 50 nt 3' of the fruA stop codon, was
reported previously (9). At that time, no open reading
frames (ORFs) were found 5' of fruA or immediately following
fruA. Also, the small amount of sequence downstream of
fruA showed no similarity to known sequences. Preliminary
nucleotide sequence analysis of more than 1.5 kbp of additional DNA 5'
of fruA indicated that there were no ORFs encoding proteins
potentially involved in fructan metabolism or gene regulation (data not shown).
As reported previously (9), there is a stable stem-loop
structure immediately downstream of fruA. An ORF beginning
71 bp from the stop codon of fruA was identified. This gene,
designated fruB, consists of 1,560 bp and is preceded by an
RBS-like sequence, but there do not appear to be sequences 5' of
fruB with similarity to canonical promoter sequences (Fig.
1). Downstream of fruB is the
dnaK operon of S. mutans, which is driven from
its own promoter(s) (31), beginning with the hrcA
gene (Fig. 1). Consistent with an S. mutans origin,
fruB is 36.9% G+C. The deduced amino acid sequence of
fruB yields a protein with a molecular weight of 58,612 and
a pI of 5.83. FruB has an N-terminal sequence, with characteristics of
signal peptides of gram-positive bacteria (56), which could be cleaved to yield a mature polypeptide with a molecular weight of
55,462 and pI of 5.49. Unlike FruA, FruB does not have an LPXTG cell
wall-anchoring sequence (49). A BLAST search of FruB against existing databases indicated that the highest degrees of similarities were to fructosidases of bacteria and lower eukaryotes, including levanases, inulinases, and invertases, and that the levels of similarity between FruB and known fructosidases were similar to those
generally demonstrated by these proteins. For example, FruB and the
Bacillus stearothermophilus levanase SurC (37)
exhibit 25% identity (37% similarity; Blast P value,
3.3e
16), and FruB shows 28% identity (36% similarity)
to a hypothetical levanase (YveB) identified in Bacillus
subtilis (Blast P value, 7.3e24).
Generally, comparisons of fructosidases across genera reveal around 25 to 30% identity and 35 to 50% similarity. Of note, FruB was only 15%
identical (29% similar) to FruA, so it seems unlikely that FruB arose
from gene duplication. Other notable features of FruB are that the
aspartic acid residue (Asp 47 in FruB) that has been shown to be
essential for catalytic activity in the yeast invertase (46)
was present in the correct relative position in FruB (Fig.
2), as was the cysteine residue (Cys 230, apparently the only cysteine in the mature FruB protein), which has
been suggested to be important for activity of sucrases.
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FIG. 1.
Relevant nucleotide sequences and features of the
fruAB gene cluster. The nucleotide sequences of the 5'
region of fruA, the fruAB intergenic region, and
the 3' end of fruB are shown. The sequences of the
structural genes have been omitted to save space. Sequence numbering
corresponds to the original GenBank submission of the fruA
sequence (accession no. L03358 and AF093758). The fruA
structural gene begins at position 685 and ends at position 4953. The
putative RBS of fruB begins at position 5016. The
fruB start codon is at position 5027, and the structural
gene extends to position 6599. Underlined and in boldface is the
extended 10-like promoter element that drives fru
transcription. Boxed and labeled as CRE-W (weaker) and CRE-S (strong)
are the CREs described in the text. The inverted-repeat structures with
characteristics of rho-independent terminators predicted by the
Terminator program of the University of Wisconsin GCG software
(indicated by opposing dashed arrows above the sequence) are in the 5'
leader mRNA of fruA (SL1, positions 599 to 626; SL2,
positions 613 to 633) and after the fruB gene (SL4,
positions 6618 to 6644). These three inverted repeats are followed by a
T (U)-rich 9-nt sequence. There is also a stable, previously identified
inverted repeat (SL3) at the end of fruA (9)
indicated by dashed arrows above the sequence, but this is not
recognized by computer algorithms as a rho-independent terminator. The
sequence with partial similarity to RAT-like sequences (RAT; positions
595 to 619), underlined with a boldfaced dashed line, overlaps with
both stem-loop structures in the leader region. The start and stop
codons of fruA and fruB are in boldface, and the
stop codons are labeled with asterisks. 35 and 10 hrcA
indicate the promoter for the dnaK operon (31).
TIS, the transcriptional initiation site mapped by primer extension.
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FIG. 2.
GAP alignment of S. mutans FruB and B. subtilis YveB. The deduced amino acid sequences of the
fruB (top) and yveB genes were aligned by using
the GAP program of the University of Wisconsin GCG package. Vertical
lines indicate identity, and single and double dots indicate lower and
high degrees of amino acid similarity, respectively. The aspartic acid
(Asp 47) believed to correspond to that in the yeast invertase which
was shown to be involved in catalysis is in boldface, as is a highly
conserved cysteine residue (position 230) found in many fructosidases.
Overlined with dashed lines are regions in FruB that exhibit some
homology to conserved regions in fructosidases or which have some
similarity to regions conserved between S. mutans FruA and
B. subtilis SacC, which are known levanases. Asterisks
indicate a sequence which is very highly conserved among FruB, YveB,
and the known levanase of B. stearothermophilus, SurC.
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Several attempts have been made to disclose the involvement of FruB, if
any, in fructan metabolism. First, a fruA mutant containing a mini-MudE transposon was previously described (59). It is now known, from the results of Northern blotting with a DNA fragment downstream of the insertion site, that the mutation in fruA
is not polar (data not shown) and, from the use of a fruB
probe, that fruB is transcribed constitutively in the
fruA mutant at levels comparable to those observed in the
wild type when fruB is fully induced (see below). Still,
fructan hydrolase could not be detected in this fruA mutant
(59). Second, E. coli carrying the intact
fruB gene under the control of the lacZ promoter
in pUC-based plasmids had no detectable fructosidase or dextranase activities. Also, FruB lacking the signal sequence and containing an
N-terminal 6-His tag has been expressed at high levels in a soluble
form in E. coli (data not shown). Screening of several independent clones demonstrated that no levanase, inulinase, sucrase, raffinase, or dextranase activity could be detected in these
recombinants, and affinity-purified FruB did not have any of the
aforementioned activities. Third, insertional inactivation of
fruB in S. mutans with a Tcr
determinant had no effect on supernatant or cell-associated fructan hydrolase activity (data not shown). Addition of the purified, histidine-tagged FruB to culture supernates from GS-5 or the
fruB mutant, both of which produce FruA, did not alter
fructan hydrolase activity appreciably (data not shown). Finally, by
measuring total fructan hydrolase activity or by using a cat
gene-fruA promoter fusion (FCAT) (see below) in a
fruB mutant, it was found that FruB does not affect the
regulation of fruA expression detectably (data not shown).
It appears that fruB transcription may not arise from its
own promoter. First, the 5' region of fruB, including
roughly 200 bp of the 3' portion of fruA, was cloned behind
a promoterless cat gene and introduced on a shuttle plasmid
into S. mutans US100. No evidence suggesting the presence of
a functional streptococcal promoter was found (data not shown). Second,
primer extension analysis using a primer complementary to
fruB mRNA yielded multiple products, typical of results
obtained when such experiments are performed with primers internal to
bacterial operons. Third, the use of RT PCR revealed the existence of a
transcript that contains fruA and fruB sequences.
Regardless of whether cells were grown on inulin (Fig.
3A) or glucose (Fig. 3B), a product was
observed, indicating that cotranscription was not dependent on growth
in medium with inducing substrates. It was also consistently observed that bands produced by RT PCR from inulin-grown cells, as well as from
cells grown on levans (data not shown), were slightly smeared and
migrated somewhat anomalously compared to the reaction products of
cells grown on hexose. This was consistently observed and was possibly
due to carryover of some polysaccharides from cells grown on fructans.
Finally, insertion of a highly polar kanamycin resistance determinant
into fruA ablated fruB transcription (data not
shown). This information, coupled with the finding that fruB
is coordinately regulated with fruA (see below), indicates that the fruAB genes constitute an operon.
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FIG. 3.
RT PCR. Shown are ethidium bromide-stained agarose gels
of products obtained by RT PCR with primers spanning the
fruA-fruB intergenic region, using RNA from cells grown on
inulin (A) or glucose (B). RT PCRs were performed with RNA from cells
grown on different carbohydrates to determine whether the carbohydrate
source affected transcriptional readthrough and because RT PCR of
fructan-grown cells always produced a band that was slightly smeared,
perhaps from carryover of polysaccharide. The sense-strand primer
corresponded to nt 4794 to 4830, and the antisense primer corresponded
to nt 5140 to 5118 for the 0.65-kbp product or to nt 5415 to 5398 for
the 0.32-kbp product (nucleotide positions are in reference to those of
GenBank accession no. AF093758). (A) Lanes 1 and 8 are DNA size
standards (LTI). Products from the PCRs were derived as follows: cDNA
prepared from S. mutans RNA with RT (lanes 2 and 5), from
S. mutans RNA without RT (lanes 3 and 6), and from GS-5
chromosomal DNA (lanes 4 and 7). (B) Lanes 1 and 6 contain the 1-kbp
ladder (LTI). Products from the PCRs were derived as follows: from GS-5
chromosomal DNA (lane 2), from cDNA prepared from S. mutans
RNA with RT (lane 3), and from S. mutans RNA without RT
(lane 4), Lane 5 contains a no DNA-no RNA-no RT control.
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Expression of fru is regulated by the carbohydrate
source.
We previously reported that fruA expression was
poor in cells grown on BHI medium containing glucose (10,
13). To determine if fruA expression is controlled by
the carbohydrate source, cells were cultured in medium containing
levan, inulin, sucrose, or fructose. Fructan hydrolase activity in the
supernatant (Table 1) and cell-associated
(not shown) fractions from mid- to late-exponential-phase cultures
(OD600 
0.6 to 0.7) was measured. FruA activity was found to be optimal when cells were grown on fructan polymers, and
activity was also higher when sucrose was provided as the growth
carbohydrate. In all cases, levan and inulin were equally efficient at
inducing fruA. Conversely, fructan hydrolase activity was
low in cells grown on readily metabolizable hexoses. Cells that were
grown on mixtures of levan and glucose, or levan and fructose, were
found to have levels of FruA activity comparable to those found when
only hexoses were used for growth (Table 1). However, when cells were
cultured on mixtures of levan and the slowly metabolized sugar alcohol
glucitol, induction was seen. Cell-associated activity paralleled that
of the supernatant FruA activity in all cases (data not shown).
Therefore, fruA expression requires induction and is subject
to carbon catabolite repression (CCR).
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TABLE 1.
Fructan hydrolase activity in supernates and CAT specific
activities of S. mutans GS-5 grown on various
carbohydrate sourcesa
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Multiple attempts were made to obtain data on the size of the
fru transcript by Northern blotting, but definitive results could not be obtained because of substantial degradation of the RNA,
which is typical of preparations from S. mutans. To
determine whether the levels of fruA mRNA were higher under
induced conditions, RNAs from cells grown in medium with inulin,
glucose, or mixtures of the two sugars were subjected to slot blot
analysis with a probe derived from an internal portion of the
fruA structural gene. The data indicated that expression of
fruA was governed in large part at the level of
transcription (Fig. 4A). This conclusion was further supported by the results of gene fusion studies (detailed below). To see if transcription of fruB and fruA
could be coordinately regulated, RNAs from cells grown on inulin,
glucose, or both carbohydrates were probed with an internal fragment of
the fruB gene (Fig. 4B). The level of
fruB-specific mRNA increased when cells were cultured under
conditions that induced fruA expression, and the increase was of a magnitude similar to that of fruA.
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FIG. 4.
Slot blot of total RNA from S. mutans grown
under induced and repressed conditions. S. mutans GS-5 was
grown in TV medium supplemented with inulin, glucose, or both at
concentrations of 0.5%. Total RNA (1 µg) was isolated from
exponentially growing S. mutans cells, treated with
RNase-free DNase, and transferred to a membrane as detailed in the
text. Hybridizations were performed with an internal fragment of either
fruA (A) or fruB (B) as indicated in Materials
and Methods. Inulin plus RNase samples were treated with RNase prior to
application to the membrane as a control to show that the RNAs were
free of DNA contamination.
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Mapping of the fruA transcription start site and
analysis of the 5' region of the fru operon.
The
results of one of four primer extension reactions, all yielding the
same conclusion, are shown in Fig. 5. A
transcriptional initiation site was identified 165 nt 5' of the
translational start site. Consistent with the results of slot blot
analysis, the primer extension signal was markedly stronger in cells
grown on fructans than in those grown on glucose. Examination of the sequences flanking the proposed transcriptional start site revealed a
10 sequence of TATACT, which compares well with the
consensus TATAAT of 
70-type promoters.
However, the sequence (GGATGGAAG) located 10 to 19 bp
upstream of position 10 did not closely match the canonical 35
sequence (TTGACA). Further examination revealed that the
promoter best resembled extended 10 promoters, which are not unusual
in gram-positive bacteria (23) and which have been
identified and demonstrated to function in Streptococcus
pneumoniae (47). Extended 10 promoters have a
conserved extension on the 10 hexanucleotide (TaTGgTATAAT),
and some can direct transcription in the absence of a 35
sequence. The proposed extended 10 promoter of fruA matches 5' extension at all positions except the less highly conserved adenine residue (Fig. 1).
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FIG. 5.
Mapping the fruA promoter. This figure shows
the results of primer extension reactions used to map the
transcriptional initiation site for fruA. RNA was isolated
from S. mutans cells growing exponentially with fructose (F)
or levan (L) as the carbohydrate source. Reactions were performed as
detailed in the text. Adjacent to the primer extensions is a sequencing
reaction performed on pFRU1 (13) with the same primer used
in the primer extension.
|
|
Two potential catabolite response elements (CREs) with strong levels of
homology to the consensus sequence for the cis-acting sites
controlling catabolite repression in gram-positive bacteria (28) were identified at positions +2 to +15 and positions
27 to 14 relative to the transcriptional start site (Fig. 1). The CRE located at nt +2 of the fru mRNA, which had the sequence
AGATAGCGCTTACA, differed from the consensus sequence,
TGWNANCGNTNWCA, only at the first position and was designated as CRE-S
(with the S being for strong homology). The second CRE
(AGATAGCGATTTGG) overlapped the promoter and differed at the
1st, 13th, and 14th positions and thus was designated CRE-W (for weaker
homology). No other good matches with CREs were identified in or near
the fruAB gene cluster. Two overlapping stable stem-loop
structures with the potential to serve as rho-independent terminators,
particularly the downstream structure, were identified in the 5'
noncoding region of the fruA mRNA (Fig. 1). The presence of
the long leader mRNA and the terminator-like structure suggested the
possibility that induction of fruA is regulated by an
antitermination mechanism, similar to some saccharolytic operons of
B. subtilis (18). Of note, there was a region of
dyad symmetry (Fig. 1), overlapping with the potential terminators,
with 50% similarity to target sites required for antitermination of
saccharolytic operons of B. subtilis, known as
ribonucleotide antiterminator (RAT) sequences (3).
Interestingly, this sequence is positioned in relation to the potential
terminators, particularly SL2, where established RAT sequences are
located with respect to known antiterminators in B. subtilis
(3).
Construction and regulation of
fru::cat transcriptional
fusions.
To demonstrate functionality of the fru
promoter region and to begin to explore whether transcriptional
regulation of fru was exerted through the putative
cis-acting elements identified above, the region containing
the fru promoter was fused to a promoterless cat
gene by strategies detailed in Materials and Methods and outlined in
Fig. 6. Briefly, the first set of
experiments involved a construct in which the entire promoter region
was fused to a cat gene in a manner such that
transcription and translation were driven from the cognate
streptococcal elements. The fruA::cat
cassette was cloned onto the streptococcal suicide vector pSF143
(53), which carries a tetracycline resistance determinant
that functions in streptococci. This construct was introduced into
S. mutans by transformation of naturally competent cells.
Transformants were analyzed by Southern hybridization and PCR to
confirm that integration had taken place and that the promoter-reporter
construct was intact (data not shown).
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|
FIG. 6.
Promoter fusions and deletion derivatives. Shown are
schematic diagrams of the derivatives of the 5' region of
fruA which were generated by PCRs and fused to an E. coli cat gene. The fruA promoter
(Pfru) is indicated with a labeled box. The two
CREs are indicated as shaded boxes overlapping the promoter (CRE-W) or
beginning at position +2 (CRE-S) in the mRNA. SDs (for
Shine-Dalgarno-Streptococcus) indicates the cognate RBS of
fruA, and SDE (for Shine-Dalgarno-E.
coli) indicates the cognate RBS of cat. (See Materials
and Methods for details.) The lollipop-like markings indicate the
approximate position of the stem-loop structures in the fruA
leader mRNA. The 5' ends of WHFRU, FCAT, 18, and 12 promoter
fusions described in the text correspond to positions 121, 439, 552, and 581, respectively. The 3' end point of WHFRU, 12, and 18 is
at position 786, and the 3' end of FCAT constructs is at position 684 (Fig. 1). All integrated constructs were introduced into wild-type
S. mutans GS-5, and all plasmid-borne derivatives were
analyzed in US100, a recA derivative of GS-5 constructed for
this study. TIS, transcriptional initiation site.
|
|
The strain chosen for further study, FCAT, was grown in TV medium
supplemented with different sugars to ascertain the effects of the
carbohydrate source on transcriptional activity from the fruA promoter. As shown in Table 1, CAT activity was six- to sevenfold higher when cells were grown on levan, inulin, or sucrose than when cells were grown on 0.5% fructose, glucose, or glucitol. These findings were consistent with the observations that steady-state levels of fruA mRNA (Fig. 4) and primer extension signals
(Fig. 5) were increased to similar degrees under inducing conditions. CAT activity was also measured in cells grown on combinations of
sugars. When cells were grown on fructan and either glucose or
fructose, CAT activity was comparable to that found with glucose or
fructose alone, further demonstrating that fruA expression was sensitive to CCR. Comparison of CAT activity in cells grown on
glucitol alone with that of cells grown on levan and glucitol reinforced the concept that expression required an inducing substrate and that repression was occurring when a more quickly
metabolized compound, i.e., fructose or glucose, was present
at 0.5%. Essentially identical results were obtained with strain
WHFRU, which included additional sequences 5' and 3' of those in
FCAT and in which translation was driven by an E. coli RBS
(Table 1). Thus, the use of cat with the E. coli
RBS, which differs from the fruA RBS at only one position,
would be suitable for 3' deletion analyses (detailed below).
Deletion analysis of the 5' region of the fru
operon.
To begin to investigate the role of the putative
cis elements, identified above, in the regulation of
fruA expression, fruA promoter deletion
constructs were obtained by PCR (Fig. 6). These deletion constructs
were fused to a cat gene containing an E. coli
RBS and cloned into pDL278. These constructs were introduced into
S. mutans US100 (S. mutans GS-5 recA)
by competent transformation. The strains were grown in TV medium with
various carbohydrate sources, and CAT activities were measured.
When strains carrying the plasmid-borne, full-length promoter and
terminator structures were assayed for CAT activity after growth on a
number of different carbon sources, enzyme levels were found to be only
marginally above the background obtained with strain DLCAT
(31) (data not shown). Interestingly, strains WHFRU and
DLFCAT grew very poorly (tg, >4 to 6 h
versus 45 to 90 min for the wild type) with fructans as the sole carbon
source. Western blot and enzyme-linked immunosorbent assay analyses
(data not shown) were performed with a FruA-specific antiserum
(13) raised against supernatant proteins prepared from
S. mutans US100 or that strain carrying either pDL278 or the
full-length promoter-cat fusions. Notably, when the promoter
region was present in multiple copies (the copy number of pDL278 has
been estimated to be around 20 to 30 [14a]), S. mutans produced barely detectable levels of FruA protein,
suggesting the possibility of titration of a trans-acting
factor required for efficient induction of fruA. Two 5'
deletion mutants (Fig. 6) in which the putative promoter was deleted
but the potential terminators were left intact were created. No CAT
activity could be measured in these constructs, the ability to grow
well on fructan substrates was restored, and FruA production was
detectable at near wild-type levels in these strains (data not shown).
These results, supported by additional data detailed below, indicate
that the 5' deletions simply ablated promoter activity.
Specifically, a 3' deletion mutant was made in which the promoter, and
sequences 5' of it, was left intact and the putative terminator and
CRE-S element were deleted, and the construct was fused to
cat with an E. coli RBS (Fig. 6). Levels of CAT
activity expressed from this construct, pDLCRE, were increased
dramatically over those of any of the integrated, full-length
constructs, and cells grew as well on fructan polymers as did the wild
type. Cells grown on fructans exhibited 700 to almost 10,000 times more
CAT activity than cells containing either the plasmid-borne or the integrated, full-length promoter fusions, depending on the growth carbohydrate (averages ± standard deviations, 1,919 ± 470, 1,662 ± 98, and 2,170 ± 545 nmol of chloramphenicol
acetylated/min/mg of protein for cells grown on inulin, glucose, or a
combination thereof, respectively). The presence of the fusion in
multiple copies likely results in higher levels than would be seen if a single copy of the construct were present. Importantly, unlike the 6- to 10-fold differences between induced and repressed activities, cells
cultivated with glucose as the sole carbohydrate source did not produce
levels of CAT statistically different from those of cells grown on
inulin. Collectively, these results support the theory that the
ascribed promoter (Fig. 1 and 5) is functional and that CRE-S is likely
involved in catabolite repression of fruA. The lack of
induction by fructans in strains carrying pDLCRE strongly suggested
that binding of DNA 3' to the promoter was not responsible for the
titration effect noted above, since the 5' deletions had no effect on
FruA synthesis or growth on fructans. Thus, transcription of the 5'
region, including the stem-loop structures, must occur to elicit
titration. A recent examination of the sequence data available through
the S. mutans genomic sequencing project being conducted at
the University of Oklahoma revealed the presence of a LicT/SacY/SacT
homologue in S. mutans UA159, supporting the hypothesis that
genes for antiterminators are present and could function in the
regulation of genes in this organism. Also, it has been determined that
the 5' regions of fruA in GS-5 and UA159 are essentially
identical (57a, 59).
Attempts were made to show definitively that the stem-loop structures
in the 5' untranslated region might function as terminators; however,
two major technical obstacles were encountered. First, a construct,
designated T, which contained the promoter and CREs but harbored a
3' deletion that eliminated the stem-loop structures could not be
stably maintained in any of the gene fusion constructs that were tried,
apparently due to intramolecular recombination. Second, as indicated
above, the introduction of constructs containing the functional
promoter and terminator clearly caused a titration effect, so
attempting to analyze the role of the proposed terminator by using
promoter fusions on a multicopy plasmid was not appropriate. Efforts to
introduce these constructs into the chromosome of S. mutans
in single copy are under way, but some problems with stability of the
vector and recombinant constructs are evident.
| |
DISCUSSION |
For S. mutans, and probably other oral bacteria,
fructans appear to serve primarily as storage compounds that can be
used when exogenous sources are exhausted (7). Therefore, it
should be beneficial to the organisms to separate fructan synthesis and catabolism temporally, postponing breakdown of the polymers until other
energy sources are exhausted. S. mutans can enhance
expression of FTF when substrate is available, and potentially the
bacteria can release the enzyme to where it can access sucrose and
convert it to polymer within the biofilm matrix (12, 27, 34,
58). Previously, we demonstrated that low pH favored association
of the FruA enzyme with the cell surface, a mechanism that could potentially restrict the access of the hydrolase to fructans when exogenous carbohydrate is present in excess (11). Although
it might be possible to optimize fructan utilization simply by
producing the hydrolase constitutively, albeit in smaller quantities
than the synthase, the data presented here indicate that expression of
fruA is tightly regulated.
It appears that fruA is actually the first gene in a
two-gene operon, and there appear to be no additional genes in the
fru cluster. A fairly thorough analysis of fruB
mutants and heterologously expressed FruB proteins has failed to
disclose the role of this protein. Of note, our inactivated
fruB construct was used to insertionally inactivate that
gene in wild-type S. mutans V403, which produces FruA and an
inulinase, as well as in a fruA mutant of V403. Strain V403
retained inulinase activity after fruA inactivation
(33), but fruB ablation had no discernible effect
on fructan metabolism (38a). That observation, coupled with
the finding that a full-length FruB protein can be produced in E. coli, indicates that it is unlikely that fruB is a
cryptic version of the inulinase gene of V403. Efforts to understand
the function of FruB are continuing.
The data presented here show that levans, inulins, and sucrose can
induce expression of fruA. Interestingly, biochemical
characterization of FruA indicates that this enzyme functions
exohydrolytically, releasing only fructose from the polymers, and that
digestion of fructans proceeds to completion (13). Since it
is unlikely that fructans directly induce expression, and since
oligomers of fructans are not produced, it is likely that fructose, at
nonrepressing concentrations, is the inducing molecule. In our initial
characterization of FruA from S. mutans GS-5
(13), we confirmed results by Jacques et al. (30)
which indicated that much higher levels of fructanase production could
be achieved in cells in steady-state continuous culture at low growth
rates (D = 0.075 h1; tg = 9.24 h) with fructose as the limiting carbohydrate than could be
achieved in batch cultures (13). Consistent with this hypothesis, fructose is the inducer of the B. subtilis
levanase, encoded by sacC (17), and high levels
of fructose are repressive through CcpA- and PtsI-dependent pathways
for CCR exerted through CREs and the transcriptional activator LevR,
respectively (41). However, there are significant
physiologic differences between S. mutans and B. subtilis that have precluded proving that fru expression is inducible by fructose. Specifically, showing induction of
levanase expression by fructose by growing B. subtilis cells on a base medium with minimal amounts of fructose has been fairly straightforward. However, this has not been possible with S. mutans since its growth is entirely dependent on substrate-level
phosphorylation and fru transcription is exquisitely
sensitive to CCR.
The results of primer extension studies and functional studies using
gene fusions are consistent with the ascribed location of the
fru promoter. The promoter is most like those of the
extended 10 type. Identification of a long leader mRNA containing
terminator-like structures which overlap with a possible RAT sequence
(3) indicates that fruA expression could be
controlled by antitermination, probably in a manner similar to
established antitermination models for saccharolytic genes, including
the sacPA and sacB genes of B. subtilis (18) and the bgl operon of E. coli (26). Notably, regulation of the levanase of
B. subtilis (SacC), which has substantial functional
similarity to FruA, is not governed by antitermination; instead,
sacC transcription is controlled primarily by the
transcriptional activator LevR (19), whose DNA binding
activity is modulated via phosphorylation by components of a
fructose-specific phosphotransferase system (PTS), encoded by
levDEFG (40). Results presented in this
communication do not support the theory that transcriptional activation
of fru is a control point for induction. Also, S. mutans chromosomal DNA has been probed with levDEFG and
levR probes (kindly provided by I. Martin-Verstraete) under
low-stringency conditions, and no evidence of sufficiently similar
sequences was found (data not shown). Similarly, although weak
similarities to the levDEFG genes, which are similar to PTS
components, can be found, there is no evidence for a LevR-like protein
in the albeit-incomplete genomic sequence. Therefore, the existing data
are most consistent with an antitermination mechanism for induction of
fru. In B. subtilis, antitermination of certain
sac genes is governed by the antiterminator proteins SacY
and SacT, whose activities are modified via phosphorylation by PTS
components (29, 54). The finding that an ORF with a very
high degree of similarity to the SacY and SacT antiterminators is
present in S. mutans supports the hypothesis that
antitermination is involved in fru induction.
There are two identifiable CREs near the fruA promoter,
located at the transcription initiation site and also partially
overlapping the promoter. CREs have been shown to be bound by a global
regulator of CCR, CcpA, which was originally cloned from B. subtilis (24). CcpA is a repressor of CCR-sensitive
genes that appears to be present in many gram-positive bacteria
(35) and which has as a corepressor a form of HPr that is
phosphorylated at serine residue 46 (32). Clearly, deletion
of the more highly conserved of the two CREs, CRE-S, which differs from
the consensus sequence at the first position, essentially eliminates
CCR. Consistent with this, Weickert and Chambliss (57) have
shown that mutation of the first nucleotide in the B. subtilis amylase CRE has little effect on CCR. Notably, mutation
of position 13 of the amylase CRE largely alleviated CCR, and thus
CRE-W, which differs in the 1st, 13th, and 14th positions, may not be
an effective cis element in the absence of CRE-S. Although
other catabolite control proteins have been found recently (14,
42), and CCR can occur by direct phosphorylation of specific
regulatory elements (41), the findings that sequences near
the fruA promoter adhere closely to the consensus for CREs
and that deletion of CRE-S alleviates CCR argue that a major control
point for CCR of fruA expression could be through binding of
a CcpA homologue which is present in S. mutans (1, 51). Interestingly, it is known that Bacillus CcpA can
bind in a cooperative fashion to two target CREs located in the
xyl operon of Bacillus megaterium
(22), so perhaps the two CREs in close proximity to the
fru promoter act as target sites for binding and
dimerization of CcpA.
In summary, the fruAB gene cluster of S. mutans
GS-5 is highly regulated by the carbohydrate source as well as its
availability, mediated through CREs known to be bound by CcpA-like
proteins, as well as potentially by transcriptional antitermination
exerted through elements in the leader region 5' to the fruA
structural gene. On the basis of recent work with B. subtilis (14, 29, 41, 42, 54), it would be surprising
if there were not other networks for controlling fruA
expression in response to carbohydrate availability. The demonstration
of tight transcriptional regulation of fru expression,
coupled with our findings on posttranslational control of localization
of FruA (11), supports the idea that S. mutans
has evolved multiple mechanisms for governing the accumulation and
utilization of fructan exopolymers. Future studies oriented toward
characterizing the trans-acting factors and other components involved in fru regulation should provide valuable
information about carbohydrate and polysaccharide metabolism in the
oral streptococci and other lactic acid bacteria.
We thank Don Wexler, Earl Albone, and Christine Hervas for
assistance with the generation of promoter constructs and preliminary regulation studies.
This work was supported by grant DE 12236 (to R.A.B.) from the National
Institute of Dental and Craniofacial Research.
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Abstract
Introduction
