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Journal of Bacteriology, November 1998, p. 5769-5775, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Transcriptional Regulation of the
Streptococcus salivarius 57.I Urease Operon
Yi-Ywan M.
Chen,1,2
Cheryl A.
Weaver,1
David R.
Mendelsohn,1 and
Robert
A.
Burne1,2,*
Center for Oral
Biology1 and
Department of Microbiology
and Immunology,2 School of Medicine and
Dentistry, University of Rochester, Rochester, New York 14642
Received 29 May 1998/Accepted 26 August 1998
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ABSTRACT |
The Streptococcus salivarius 57.I ure
cluster was organized as an operon, beginning with
ureI, followed by ureABC (structural genes) and
ureEFGD (accessory genes). Northern analyses revealed transcripts encompassing structural genes and transcripts containing the entire operon. A 
70-like promoter could be
mapped 5' to ureI (PureI) by primer extension analysis. The intensity of the signal increased when cells were grown
at an acidic pH and was further enhanced by excess carbohydrate. To
determine the function(s) of two inverted repeats located 5' to
PureI, transcriptional fusions of the full-length promoter region (PureI), or a deletion derivative
(PureI
100), and a promoterless chloramphenicol
acetyltransferase (CAT) gene were constructed and integrated into the
chromosome to generate strains PureICAT and
PureI100CAT, respectively. CAT specific activities of
PureICAT were repressed at pH 7.0 and induced at pH 5.5 and
by excess carbohydrate. In PureI100CAT, CAT activity was
60-fold higher than in PureICAT at pH 7.0 and pH induction
was nearly eliminated, indicating that expression was negatively
regulated. Thus, it was concluded that PureI was the
predominant, regulated promoter and that regulation was governed by a
mechanism differing markedly from other known mechanisms for bacterial
urease expression.
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TEXT |
Ureases are multisubunit enzymes
requiring Ni2+ for catalytic activity. Several bacterial
urease gene clusters have been isolated and characterized, and
similarities in the organization of the clusters between species have
been demonstrated (17, 18). Most bacterial ureases consist
of three subunits, 
, 
, and 
, encoded by ureC,
-B, and -A, respectively (18),
although exceptions exist (10). The assembly of a
catalytically active urease also requires ureE,
-F, -G, and -D, known as accessory
genes, which encode proteins required for the productive incorporation
of Ni2+ into the metallocenter within the active site. In
some cases, high-affinity nickel transporters, for instance, NixA of
Helicobacter pylori (16), are known to be
required for optimal urease activity. Additional genes, such as
ureI of H. pylori and ureH and
ureI of Bacillus sp. strain TB-90, have been
identified (15), but the function(s) of these gene products
in urease biogenesis is not well defined. However, UreH from
Bacillus has homology with the high-affinity nickel
transporter of Alcaligenes eutrophus, HoxN (31);
thus, it has been proposed that UreH is involved in energized nickel
uptake in conjunction with UreI.
The expression of bacterial urease genes is regulated by different
mechanisms (7). Few known bacterial ureases are expressed constitutively, whereas most are regulated by environmental conditions. For example, urease expression in Klebsiella pneumoniae and
Klebsiella aerogenes is activated only under
nitrogen-limiting conditions (8, 14, 19), and expression in
Proteus mirabilis is induced by urea and is mediated by the
positive transcriptional regulator UreR (20). Information
regarding the expression of ureases of oral bacteria is just beginning
to accumulate. Among the species of oral bacteria that have been
identified as ureolytic, Streptococcus salivarius is
believed to be a major contributor to total oral ureolysis
(25). In contrast to studies of other known mechanisms for
urease expression, previous studies in our laboratory and in
others demonstrated that urease expression in S. salivarius 57.I is regulated by the environmental pH
(4, 27), growth rate, and carbohydrate availability
(4). At a neutral pH, expression is almost
completely repressed. Induction occurs when cells are grown at an
acidic pH, and expression is further enhanced by excess carbohydrate
and higher growth rates. Furthermore, based on the results of Northern
blot analysis measuring ureC-specific mRNAs from cells grown
at various pH values and in conditions with limited or excess
carbohydrate, it was found that the induction is regulated, at least in
part, at the transcriptional level (4).
Our initial attempts to isolate the urease genes from S. salivarius 57.I resulted in the cloning of a partial gene cluster that contained the 3' end of ureI followed by
ureABCEFGD (5). Despite the lack of the 5' end of
the cluster, Streptococcus gordonii DL1, a
nonureolytic oral microorganism, expressed urease activity when
harboring this partial ure cluster on a
moderate-copy-number plasmid (pMC17) (Fig.
1; Table 1)
and when the growth medium was supplemented with NiCl2
(5). The purpose of this work was to identify
additional genes that may be involved in urease biogenesis, to
investigate the transcriptional organization of the ure
cluster of S. salivarius 57.I, and to explore the basis
for differential expression of ure genes in response to pH
and carbohydrate availability.
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FIG. 1.
ure cluster and the 5' flanking region on the
S. salivarius 57.I chromosome. A restriction
endonuclease map of the chromosome containing the ure
cluster and the 5' flanking region is shown on the top line. The limits
of the DNA sequence shown in Fig. 2 are indicated by vertical arrows.
The relative location and the direction of transcription of each ORF
are indicated by horizontal arrows. The molecular mass in kilodaltons
of each gene product is shown below each gene. The locations and
orientations of primer pairs used in RT-PCR are indicated by arrows
immediately under the restriction map. The
Sau3A-XbaI region from pMC12, used as a probe for
identifying pMC32, is indicated in a hatched box. The region used for
the construction of pCW45 and pMC77 (see below) for integration is
indicated in a shaded box within the restriction map.
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Isolation and nucleotide sequence analysis of the 5' portion of
ureI and the flanking region from S. salivarius 57.I.
A 7.8-kbp Sau3A fragment
containing the 3' portion of ureI and complete
ureABCEFGD has been previously described (5). No genes involved in urea metabolism were found within 1.5 kbp 3' to the
ure cluster, and a putative Rho-independent terminator was
identified 120 bases 3' to the stop codon of ureD, the last gene in the cluster. To obtain the complete ureI and other
genes potentially involved in urease biogenesis, total chromosomal DNA was isolated from S. salivarius 57.I and digested to
completion with XbaI. DNA fragments were separated on
agarose gels, transferred to nitrocellulose, and hybridized at high
stringency (12) to a radiolabeled
Sau3A-XbaI fragment, approximately 1.0 kbp in
size, which contained the 3' portion of ureI (Fig. 1). An
approximately 6.0-kbp fragment was subsequently identified (data not
shown). To isolate this fragment, a subgenomic DNA library was
constructed in pSU21 (2) and screened with the same probe,
as described above. The resulting chimeric plasmid was designated
pMC23. To maintain stably the XbaI fragment in
Escherichia coli, it was necessary to subclone the insert
onto a low-copy-number vector, pDL290 (10a), which generated
pMC32. For convenience, all strains and plasmids used in this study are
listed in Table 1.
The complete nucleotide sequences of both strands of the
XbaI fragment were obtained, and the sequences of
ureI and the 5'
flanking region are presented here. The
ure cluster began with
an open reading frame (ORF) of
513 bp, which had homology with
H. pylori ureI and thus was
designated
ureI (Fig.
2). A putative
ribosome binding
site (RBS) could be found 6 bases 5' to the predicted
start codon of
ureI. The
ureI gene was predicted to encode
a protein
with an estimated molecular weight of 18,995 and a pI of 7.0.
In addition, the hydropathy plot of the deduced amino acid sequences
indicated that UreI was relatively hydrophobic, with six potential
transmembrane domains (data not shown), suggesting that UreI
could
be a membrane protein.
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FIG. 2.
Nucleotide and deduced amino acid sequences of the 5'
flanking region of the ure cluster. ORF3 and ureI
are transcribed from the opposite DNA strands; thus, the sequence of
ureI presented here is the coding strand, and the sequence
of ORF3 is the noncoding strand. The locations and orientations of
primers (PureIas-100 and PMC32-1) used to identify the
transcriptional start site of ureI and of primers used to
amplify PureI and PureI100 (PureIs,
PureIde12, and PureIas) are indicated by
horizontal arrows. The transcriptional start site of ureI,
determined by primer extension analysis, is indicated by a vertical
arrow, and the corresponding  10 and 35 regions are overlined. The
sequences of the inverted repeats 5' to PureI are shaded.
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Translation of the nucleotide sequences of the
XbaI
fragment revealed three additional ORFs located 5' to the
ure cluster.
ORF1 and -2 were transcribed in the same
direction as the
ure cluster, and ORF3 was transcribed in
the opposite direction (Fig.
1). Proposed RBSs were found in the
appropriate positions 5' to
all ORFs. ORF1, 2,094 bp in size,
encoded a protein with a calculated
molecular weight of 78,431 and an estimated pI of 8.9. A high
degree of homology was observed
between ORF1 and ATP-binding cassette
transporters. ORF2, located 109 bp 3' to ORF1, was 330 bp and
encoded a protein with a calculated
molecular weight of 12,717
and an estimated pI of 9.6. Based on the
hydropathy plot, ORF2
may encode a membrane protein; however, only low
levels of similarity
were observed between the ORF2 product and other
known proteins.
ORF3, 1,392 bp in size, was transcribed in the
direction opposite
to the transcription of ORF1 and -2, and it encoded
a protein
with a calculated molecular weight of 52,393 and an estimated
pI of 9.7. Due to the proximity of these three ORFs to the
ure cluster and their characteristics, the possibility of
their involvement
in optimal urease expression, perhaps through
Ni
2+ uptake, is currently under investigation.
Operonic arrangement of the S. salivarius 57.I
ure cluster.
To analyze the transcriptional
organization of the ure cluster and potentially identify a
transcript(s) that is induced at a low environmental pH, Northern blot
analyses were performed with batch-grown cells at pH 7.0, 6.0, and 5.0, and ureI-, ureC-, and ureDG-specific
mRNAs were examined (Fig. 1 and 3A).
Total RNA was isolated according to the method of Putzer et al.
(21) with modifications. Briefly, cells were cultured in
brain heart infusion (BHI; Difco, Detroit, Mich.) containing 50 mM
potassium phosphate buffer, pH 7.5, in BHI alone, or in BHI which had
been adjusted to pH 5.5 by addition of 2 N HCl to mid-log phase, at which point the cultures were at approximately pH 7, 6, and 5, respectively. Cells were harvested, washed once with 10 mM sodium phosphate buffer, pH 7.0, and then resuspended in 1/40 of the original
culture volume in 50 mM Tris-10 mM EDTA, pH 8.0. In 2.0-ml screw-cap
microcentrifuge tubes, 0.5 g of glass beads (0.1-mm diameter),
500 µl of concentrated cell suspensions, 500 µl of phenol-chloroform (5:1, pH 4.7; Ambion, Austin, Tex.), and 100 µl of 10% sodium dodecyl sulfate were added. Cells were then
subjected to mechanical disruption by homogenization in a Bead Beater
(Biospec Products, Inc., Bartlesville, Okla.) for a total of 40 s
at 4°C. The aqueous phase was first extracted with an equal volume of phenol-chloroform four to five times, followed by extraction with chloroform-isoamyl alcohol (24:1) alone. Total cellular RNA was then
precipitated with 1/10 volume of 3 M Na acetate, pH 6.0, and 2 volumes
of ice-cold 99% ethanol. Northern blot analysis was performed with
0.7% formaldehyde-agarose gels (22), and hybridization
conditions were as described previously (1).
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FIG. 3.
(A) Northern blot analysis of ure-specific
messages. Ten micrograms of total cellular RNA from cultures at each pH
level was probed with a ureI-specific probe (a), a
ureC-specific probe (b), and a ureDG-specific
probe (c). (B) PCR products generated from RT-PCR. 1% of total cDNA
generated by RT-PCR from each RNA sample was amplified with specific
primers (Fig. 1), and 10% of the PCR products were run on a 0.8%
Tris-borate-EDTA gel. Some PCR products were generated with a primer
pair specific for the ureIA intergenic region (a), and
others were generated with a primer pair specific for the
ureCE intergenic region (b). RT was included in some
reactions (+RT), but not in control reactions which were carried out
identically to the experimental samples (RT). In other control
reactions PCRs were used to amplify the target region from
S. salivarius 57.I chromosomal DNA under the same
conditions (57.I). The 100-bp DNA ladder was used as the molecular
weight marker.
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Two species, approximately 6.8 and 2.7 kb, were identified with a probe
specific for
ureI. The intensity of both signals increased
when the culture pH became acidic, and the highest levels were
observed
in cells grown at pH 5.0. Furthermore, under all growth
conditions, the
smaller transcript was more abundant than the
larger one. Extrapolating
from the estimated sizes of RNA transcripts
and the known sizes of
the urease genes, the two transcripts presumably
corresponded to
ureIABCEFGD and
ureIABC, respectively. When
total
cellular RNA was probed with a
ureC-specific probe,
close examination
revealed four fragments, 6.8, 6.3, 2.7, and 2.2 kb,
presumably
corresponding to
ureIABCEFGD,
ureABCEFGD,
ureIABC, and
ureABC,
respectively. However, due to degradation of the mRNA, which is
commonly observed with RNA from oral streptococci, it was not
possible
to definitely show by Northern analysis that the transcripts
could
arise from two promoters. The intensities of the signals
increased when
the growth pH was acidic. However, it was difficult
to determine
whether all transcripts were enhanced at an acidic
pH or only
transcripts arising from the promoter 5' to
ureI
(
PureI).
In agreement with the above results, only the
larger two transcripts,
6.8 and 6.3 kb, were observed when a
ureDG-specific probe was
used. Although the signals
were not as sharp as those seen in
ureI- and
ureC-specific messages, increases in intensity of signals
were observed in cells grown in acidic media. We did not
observe
any transcripts of the appropriate size for
ureEFGD,
suggesting
the absence of a functional promoter 5' to the accessory
genes.
In addition, all attempts, including the use of primer
extension
analysis and promoterless reporter fusions, failed to
identify
functional promoters within the
ureCE intergenic
region.
To confirm that transcription could extend through the
ureIA and
ureCE intergenic regions, the
presence of the larger transcripts
was verified by reverse
transcriptase (RT) PCR. cDNA was synthesized
from 10 µg of total RNA
isolated from cells grown under different
conditions with Moloney
murine leukemia virus RT and was further
amplified by PCR with random
hexamers according to standard procedures
(
9). Negative
controls included reactions in the absence of
RT. cDNAs spanning the
ureIA and
ureCE intergenic regions were
detected
by PCR with primer pairs with appropriate sequences.
In both cases, PCR
products with appropriate sizes (Fig.
3B) and
correct sequences (data
not shown) were obtained, further supporting
the operonic
arrangement of the
ure cluster. No products were
seen in
control reactions that were designed for detecting chromosomal
DNA
contamination.
It has been proposed that, after activation of the urease
apoenzyme, the accessory proteins are released from the holoenzyme
and are recycled for the purpose of assembling another catalytically
active urease (
18). Consequently, the quantity of structural
proteins (UreABC) could be greater than that of accessory proteins
(UreEFGD) at any given time. Results obtained with Northern analysis
are consistent with the theory that transcripts containing the
accessory genes are less abundant than those carrying only the
structural genes. Although the molecular basis for the generation
of
the smaller transcripts (
ureIABC and
ureABC) is
not clear,
a strong stem-loop structure (
G =
34 kcal/mol)
followed by a
string of uridine residues was located 11 bases 3' to the
stop
codon of
ureC. We did not observe any transcripts with
sizes corresponding
to
ureEFGD; thus, it is not likely that
the smaller transcripts
resulted from posttranscriptional
processing. In preliminary half-life
studies, it appears that the two
smaller transcripts have half-lives
roughly three times longer than
those of the larger transcripts,
which is not sufficient to account for
the disparity in the amounts
of the two sets of transcripts under fully
induced conditions.
Therefore, it seems likely that preferential
termination occurs
in the
ureCE intergenic region, which
gives rise to a higher proportion
of
ureIABC and
ureABC transcripts compared with
ureIABCEFGD and
ureABCEFGD transcripts.
Localization of PureI.
Based on the results
obtained from Northern blot analyses and the fact that ureI
is the 5'-most gene in the ure cluster, it was hypothesized
that PureI was differentially regulated in response to
the environmental pH and carbohydrate availability. To map the putative
location of PureI, the 5' end of the transcript was determined by primer extension analysis. Total cellular RNAs
from cells growing at steady state in continuous culture with a
dilution rate of 0.3 h
1 (generation time 
2.3 h),
at pH 7.0 or 5.5 under carbohydrate-limiting conditions (10 mM
fructose) or at pH 5.5 under excess carbohydrate conditions (200 mM fructose), were isolated. Cells were grown for at least 10 generations at any single set of growth parameters before cultures were
defined to be at steady state, where the specific growth rate was equal
to the dilution rate of the vessel (28). Two primers,
containing antisense sequences of ureI, were used: primer
PureIas-100, 5'-TCAAACCCAAACCACCCG-3', and
primer PMC32-1, 5'-CCCTGTACAAGCTCCAT-3', were
located 106 and 148 bases 3' to the start codon, respectively
(Fig. 2). Products extended from each reaction were analyzed on a 6%
polyacrylamide gel along with a DNA sequencing reaction with the same
primer. A signal, 22 bases 5' to the translational start site of
ureI (Fig. 4), was
consistently observed with either primer under all growth conditions.
This transcriptional initiation site could be mapped to a
70-like promoter sequence located at an appropriate
distance (Fig. 2). Furthermore, the intensity of the signal was
greater with RNAs isolated from pH 5.5-grown cells than with those
from pH 7.0-grown cells. The strongest signal was observed with RNAs
isolated from cells grown at pH 5.5 with excess carbohydrate,
suggesting that PureI is sensitive to both an acidic pH and
carbohydrate concentrations.
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FIG. 4.
Primer extension analysis of PureI. Total
cellular RNA of S. salivarius 57.I from steady-state
cultures grown in a chemostat were used. Radiolabeled primer PMC32-1
was incubated with the RNA, and the corresponding DNA was synthesized.
The same primer was used to prime dideoxy sequencing reactions with
plasmid pMC32 as a template. Lanes 1 and 2 show total RNA isolated from
pH 7.0 and pH 5.5 cultures, respectively, with 10 mM fructose. Lane 3 shows total RNA isolated from a pH 5.5 culture with 200 mM fructose.
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Based on the results of Northern blot analysis and the
NiCl
2-dependent urease expression in recombinant
streptococcal and
E. coli strains harboring
ureA
to -
G (
5), the possibility exists
that there is a
functional promoter 5' to
ureA (
PureA). Our
preliminary
study using a
PureA-cat fusion, in which a
400-bp fragment beginning
5' to the translational start codon of
ureA was fused to a promoterless
chloramphenicol
acetyltransferase (CAT) gene (
cat), also demonstrated
a
functional but pH-unresponsive streptococcal promoter within
this
400-bp region (data not shown). In addition, we found extremely
low
levels of urease in a strain of
S. salivarius carrying
a polar
insertion in
ureI, where the expression of the
ure operon should
have been derived solely from the
activity of
PureA (data not
shown). However, all attempts to
localize
PureA by primer extension
have failed. Multiple
signals, probably due to readthrough from
PureI, were
observed, making it impossible to determine the location
of
PureA. When these observations are taken together, we
cannot
rule out the possibility that
PureA is functional in
vivo. However,
it is quite clear that
PureA is not the
promoter used for the
differential expression of urease in response to
environmental
signals. Thus, we focused on the analysis of the dominant
promoter
in the operon
PureI.
Molecular analysis of the 5' flanking region of PureI.
Sequence analysis revealed two inverted repeats, 26 and 83 bases 5' to
the 35 region of PureI, with G values of 19 and 15
kcal/mol, respectively (Fig. 2). To determine whether these putative secondary structures could function as cis-acting
elements in response to environmental signals, two transcriptional
fusions to a promoterless cat were constructed. The
promoterless cat fragment containing an E. coli
RBS was purchased from Pharmacia (Piscataway, N.J.) as a
HindIII fragment. To assist the cloning, sequences recognized by SalI and BamHI were included 5' to
the ATG site of the cat fragment
(5'-GCG TCGACTGGATCCATGGAGAAAAAAATCACT-3'), and sequences
recognized by HindIII were included in the primer which contains the antisense sequences of the 3' end of the cat
sequences (5'-CAAGGATCCAAGCTTCGACGAATT-3'). PCRs
were used to amplify the cat fragment with the pair of
primers described above. The PCR products were initially digested with
SalI and HindIII and then cloned onto
SalI- and HindIII-digested pUC18 to obtain a
chimeric plasmid in which the cat fragment formed a
translational fusion with lacZ. The ligation was used
to transform E. coli DH10B, and selection was carried
out to obtain transformants that were resistant to 50 µg of
chloramphenicol per ml. The resulting plasmid was designated pCW24.
To assist in the construction of pMC68 and pMC71 (see below), the
HindIII site on this cat fragment was
destroyed by digesting pCW24 with HindIII, followed by
treatment with Klenow fragments and deoxynucleoside triphosphates. The
cat fragment was then released from pCW24 by SalI
digestion. The resulting fragment was subsequently cloned onto
EcoRV- and SalI-digested pGEM-5Zf(+)
(Promega) to generate pCW42.
Because of the proximity of the putative
35 region to the inverted
repeat immediately on the 5' side, it was difficult to
design primers
for the construction of a deletion derivative completely
lacking both
inverted repeats. Consequently, the intact promoter
(
PureI)
and the deletion derivative lacking 1.5 inverted repeats
(
PureI100) were amplified by PCRs with the primer pair
P
ureIas
and P
ureIs and the primer pair
P
ureIas and P
ureIde12, respectively
(Fig.
2). To
facilitate the construction with the promoterless
cat, a
BamHI site was included immediately 5' to the ATG site
of
ureI in primer P
ureIas,
5'-CACCTAACAT
GGATCCCTCCTAAG-3'. Primers
P
ureIs (5'-GGCGACAATCAGTCCCTTAAT-3') and
P
ureIde12 (5'-T
AAGCTTGACTAATATGTAAATG-3'),
containing a
HindIII site for cloning, were
located 535 and 80
bases 5' to the ATG site of
ureI,
respectively. The PCR products
of
PureI and
PureI100 were initially cloned onto pCRII (Invitrogen,
Carlsbad, Calif.), and the nucleotide sequences were determined.
The correct products were then cloned onto
BamHI- and
HincII-digested
pCW42, selecting for resistance to
chloramphenicol. Because of
the lack of an alternative integration
vector, a 2.0-kbp fragment
(Fig.
1) located 5' to the inverted repeats
was ligated to both
constructs at the
HindIII site to
generate pMC68 and pMC71, respectively.
To facilitate the integration
of the reporter constructs into
the
S. salivarius
57.I chromosome, the
ureI promoter-
cat fusions
were then released from pMC68 and pMC71 and subcloned onto pSF143
(
29) in
E. coli, selecting for transformants
resistant to 5
µg of tetracycline per ml and 50 µg of
chloramphenicol per ml.
The resulting plasmids were designated pCW45
and pMC77, respectively.
Both plasmids were introduced into
S. salivarius by
electroporation according to the guidelines of Caparon and Scott
(
3)
with the following modifications. Briefly, an overnight
culture
of
S. salivarius 57.I grown in Todd-Hewitt
broth (Difco) and containing
0.2% yeast extract (THY) and 300 mM
L-threonine was diluted 1:20
in fresh medium
containing the same concentration of
L-threonine.
Cultures
were incubated at 37°C in a 5% CO
2 atmosphere until the
optical density at 600 nm reached 0.2. Cells were kept on ice
for
10 min prior to harvesting by centrifugation at 4°C. Cells
were
washed twice with an equal volume of ice-cold electroporation
medium
(272 mM glucose, 1 mM MgCl
2 [pH 6.5]) and then
resuspended
in 1/15 of the original culture volume in ice-cold
electroporation
medium. Concentrated cell suspensions were kept on ice
for at
least 45 min prior to electroporation. Aliquots (40 µl) of the
cells were mixed with 200 ng of plasmid DNA and then transferred
into a
chilled 0.1-cm Gene Pulser electroporation cuvette. Negative
controls
included cells with no added DNA. Electroporations were
carried out at
1.8 kV, 25 µF, and 200
. Cuvettes, containing
cells and DNA, were
kept on ice for 2 min after electroporation.
The cell suspensions were
subsequently recovered with 1 ml of
THY plus 10 mM glucose at 37°C in
a 5% CO
2 atmosphere for 2 h.
Cells were then
concentrated and plated on BHI agar supplemented
with 3 µg of
tetracycline per ml. All plates were incubated at
37°C in a 5%
CO
2 atmosphere overnight. Colonies were usually visible
in
18 to 24 h. The tetracycline-resistant transformants,
strains
PureICAT and
PureI100CAT, were further confirmed by
Southern
blot analysis with a tetracycline-specific probe (data
not shown).
Because of the nature of single-cross integration, both
strains
were predicted to possess a wild-type promoter 5' to the
ure cluster
and a full-length promoter, or the
deletion derivative, 5' to
cat. To confirm the presence and
configuration of the constructs,
a primer located 100 bases 3' to the
ATG and containing the antisense
sequences of
cat and a
primer located 5' to the
HindIII site on
the wild-type
chromosome were used to amplify the chromosomal
region of strains
PureICAT and
PureI100CAT. The PCR products
were subsequently cloned onto pCRII and subjected to
sequence
analysis to confirm that the junction of the promoter
region and
the
cat gene was intact and to ensure that
the deletion of the
inverted repeats 5' to
cat
had occurred when desired.
To be certain that the expression observed in
PureI100-CAT was due only to
PureI and
not to promoters 5' to the deleted region,
the transcriptional
initiation site for the
cat gene was determined
in
PureI100CAT by primer extension analysis with a primer
100
bases 3' to the translational start site of
cat (Fig.
5). The
results indicated that the
transcriptional initiation site matched
the initiation site used in the
wild type. The expression of
cat in both strains was
determined in chemostat-grown cultures under
different environmental
conditions (Table
2). Briefly, cells
were
harvested and washed once with an equal volume of 10 mM Tris,
pH 7.8, and then resuspended in 1/40 of the original culture volume
in the same
buffer. Concentrated cell suspensions were subjected
to mechanical
disruption in the presence of an equal volume of
glass beads (0.1-mm
diameter) by homogenization in a Bead Beater
for a total of 2 min
at 4°C. The concentration of each protein
lysate was measured
by using a protein assay (Bio-Rad, Hercules,
Calif.), based on
the method of Bradford. Bovine serum albumin
served as the
standard. The rates of chloramphenicol acetylation
of each protein
lysate were quantitated by the method of Shaw
(
23). Low
levels of expression were observed in
PureICAT grown
at a
neutral pH under carbohydrate-limiting conditions (Glc
concentration
= 20 mM). Induction, approximately eightfold
increases, occurred
when the culture became acidic, with the highest
levels observed
in cells grown at pH 5.5 under excess carbohydrate
conditions
(approximately 17-fold increases). This result was
consistent
with previous observations in which the levels of the
ureC-specific
mRNAs were induced to a comparable
magnitude under similar growth
conditions (
4).
Interestingly, at a neutral pH the specific
activity in
PureI100-CAT was approximately 60-fold higher
than
that in
PureICAT. Modest induction by an acidic pH was
still observed
in
PureI-100CAT but was never more than
twofold under carbohydrate
limiting conditions. No further induction by
excess carbohydrate
was observed in
PureI100CAT.
These data indicated that
PureI was negatively
regulated and that the expression of
PureICAT was
derepressed at an acidic pH. The slight increase in activity by
an
acidic pH observed in
PureI100CAT under
carbohydrate-limiting
conditions may be due to the presence of partial
sequences of
the inverted repeat, with which the proposed repressor
might still
weakly interact.
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|
FIG. 5.
Primer extension analysis of PureI100CAT.
Total cellular RNA of S. salivarius PureI100CAT from
a steady-state culture grown at pH 5.5 with 20 mM glucose was isolated,
and the radiolabeled primer cat (5'-AATGCCTCAAAATGT-3')
was used. The DNA sequences were derived from pMC71 with the same
primer.
|
|
The regulation of
S. salivarius 57.I urease expression
by the environmental pH and carbohydrate availability, but not nitrogen
availability, is distinct from previously defined urease control
pathways, yet control of urease expression in this manner seems
particularly well suited to the environment occupied by oral
streptococci.
It has been established that oral streptococci experience
large
and rapid changes in carbohydrate availability and pH. Moreover,
it has been documented that ammonia generation from urea protects
S. salivarius against lethal acidification
(
24). Logically,
then, substantial up-regulation of urease
gene expression at a
low environmental pH and high carbohydrate
concentrations would
offer the organism a mechanism to induce synthesis
of this protective
system under conditions when the enzyme might be
needed most for
survival. High levels of ureolysis may be
especially important
for the survival of
S. salivarius
under extreme acidic conditions,
as it is considered to be less
aciduric than some oral streptococci
and lactobacilli. On the other
hand, repression of
ure expression
at a neutral pH could
avoid overproduction of ammonia and alkalization
of the environment,
which can also be lethal for ureolytic organisms
(
6).
In this study, we have completed the cloning and sequence analysis of
the
ure operon. The operon began with
ureI, followed
by the structural genes,
ureABC,
and then the accessory genes,
ureEFGD. The function of UreI
is not well defined, although it
is clearly not required for urease
biogenesis, since heterologous
expression of the
S. salivarius ureA to -
D in streptococcal hosts
yields a
urease enzyme indistinguishable from that of the parent
(
5).
Notably, we have investigated the possibility that UreI
could be
involved in a regulatory circuit governing transcription
of
ure genes in response to pH. Using a strain containing a
polar
insertion in
ureI, we found that pH responsiveness of
PureI remained
identical to that of the wild type (data not
shown), strongly
suggesting that the expression of
ureI is
not autogenously regulated,
nor is its gene product required for
pH-dependent regulation.
The expression of both CAT and urease was induced by an acidic pH
and excess carbohydrate in strain
PureICAT, in which the
transcription of both the
ure operon and
cat was driven by a full-length
PureI.
However, the magnitude of increases in CAT level was substantially
less
than that of urease. The differences are likely attributable
to the
observation that the half-lives of CAT and of urease in
oral
streptococci are dramatically different. It has been shown
that the
S. salivarius urease is extremely stable in vivo
(
26);
thus, the activity observed with each growth condition
would reflect
the combination of the amount of stable mRNA and the
accumulation
of a stable enzyme. On the other hand, the turnover rate
of CAT
in oral streptococci appears to be relatively high
(
30); hence,
the level of CAT specific activities would be
more closely related
to the level of transcription initiation.
Consistent with this,
the magnitudes of increases in the levels of
transcription initiated
by
PureI measured by primer
extension (Fig.
4) and in the levels
of
ureC-specific
messages quantitated by slot blot analysis (
4)
most closely
parallel the increases in CAT specific activity in
response to an
acidic pH and excess carbohydrate. It should also
be noted that the
level of expression observed in
PureICAT under
acidic pH and
excess carbohydrate conditions never reached the
level observed in
PureI100CAT. It is possible that other factors
may
influence the expression of
PureI100CAT, which may
have been
reflected in the apparent higher strength of
PureI100. However,
it is more likely that
PureI is not completely derepressed under
the conditions
examined and that perhaps more-acidic conditions
are needed to
fully derepress the operon.
In summary, we have established that the urease genes of
S. salivarius constitute an operon, have identified a
promoter that
is sensitive to both the environmental pH and
carbohydrate availability,
and have determined that the induction of
urease expression by
an acidic pH and excess carbohydrate was
negatively regulated.
Unlike reports of enteric bacterial ureases,
which are either
constitutively expressed or regulated by an activator,
this is
the first report to demonstrate negative control of urease
expression.
Efforts to isolate and characterize the
trans-acting factors which
bind near
PureI are
under way.
Nucleotide sequence accession numbers.
The sequence of
ureI was submitted to GenBank and assigned accession no.
AF042344. The sequences of ORF1, -2, and -3 were assigned
GenBank accession no. AF043280, AF043281, and AF043282, respectively.
| |
ACKNOWLEDGMENTS |
We thank R. G. Quivey and K. A. Clancy for critical
review of the manuscript.
This study was supported by PHS grant DE10362 from the National
Institute for Dental Research to R.A.B.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for Oral
Biology, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Phone: (716) 275-0381. Fax: (716)
473-2679. E-mail: robert_burne{at}urmc.rochester.edu.
| |
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