Department of Microbiology, Technical
University of Denmark, DK2800 Lyngby, Denmark
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INTRODUCTION |
In nature, ATP and GTP are both
derived from IMP, which is synthesized de novo from
5-phosphoribosyl-1-pyrophosphate (PRPP), glycine, glutamine, aspartate,
and C1 units by the action of 10 enzymatic reactions
(41). While the individual enzymatic steps in purine
biosynthesis appear to be similar in all bacteria, the genetic
organization and the regulation of the pur genes follow different rules in different bacteria.
In the gram-negative bacterium Escherichia coli, the purine
biosynthetic genes are scattered around the chromosome. However, the
transcription of all of these genes is repressed by a single regulatory
protein, the purR-encoded purine repressor (11, 23, 29). Binding of the E. coli PurR repressor to its
target DNA sequences (PurBox's) is stimulated by the corepressors
guanine and hypoxanthine (24, 30). In the gram-positive
bacterium Bacillus subtilis, all genes involved in the
synthesis of IMP are organized in a single transcriptional unit, the
purine operon (6). The transcription of the operon is
controlled by two independent mechanisms. Initiation is controlled by a
repressor which is encoded by the purR gene and which, at
low concentrations of PRPP, binds specifically to a DNA sequence in the
promoter region (8, 39). The purR genes from
B. subtilis and E. coli are unrelated, and the
B. subtilis protein shows a high degree of similarity with purine phosphoribosyltransferases (39), while the
E. coli protein is a classical lacI-type
repressor (34). An attenuator model for the regulation
of premature termination of transcription has been proposed
(6). The formation of an antiterminator structure between
the promoter and the structural genes has been suggested to be
prevented by the binding of an unidentified RNA binding protein in the
presence of a guanine nucleotide, thus resulting in the termination of
transcription.
Recently, the purDEK operon (26) and the
purC gene (18) were cloned from Lactococcus
lactis CHCC285, and their nucleotide sequences were determined. A
deletion analysis of the purD and purC promoters
indicated that the regulatory element is a transcriptional activator
and that the standard 
70 factor is involved
(26).
In the present study, we have isolated ISS1 transposon
mutants (21) with purine auxotrophic phenotypes. One of the
mutated genes was identified as a homolog of the purR gene
from B. subtilis. We report the characterization of the
purR::ISS1 mutant, the nucleotide sequence of the wild-type purR gene, and the identification
of the purR promoter. Moreover, we show that the
purR gene encodes an activator required for the expression
of the purine biosynthetic genes of L. lactis.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The strains used in
the present study are listed in Table 1.
E. coli cells were grown in Luria-Bertani medium
supplemented with ampicillin at 50 µg/ml and erythromycin at 150 µg/ml when required. Cultures of L. lactis were grown in
SA medium (16) supplemented with 1% glucose (GSA medium)
and erythromycin at 2 µg/ml when necessary.
Plasmids and primers.
The plasmids and oligonucleotide
primers used in the present study are listed in Tables
2 and 3,
respectively.
Transformation.
L. lactis was transformed by
electroporation (13). E. coli cells were
transformed as described before (31).
DNA isolation, manipulations, and sequencing.
Chromosomal
lactococcal DNA was prepared as described by Johansen and Kibenich
(17). The general in vitro DNA methods described by Sambrook
et al. (31) were used. DNA sequences were determined from
plasmid DNA by the dideoxy chain termination method (32) with a Thermosequenase radiolabeled terminator cycle sequencing kit
(product US 79750; Amersham LifeScience) in accordance with the
protocol of the manufacturer.
Southern blot analysis.
Southern blot analysis was performed
with GeneScreen nylon membranes (New England Nuclear) and the
digoxigenin (DIG) system (Boehringer Mannheim Biochemicals) for
colorimetric detection of hybridized products in accordance with the
protocols specified by the manufacturers.
PCR amplification of DNA.
L. lactis chromosomal DNA
was amplified by PCR with 1 µg of template DNA in a final volume of
100 µl containing deoxyribonucleoside triphosphates (0.25 mM each),
oligonucleotides (10 µM), and 2.5 U of AmpliTaq DNA polymerase (The
Perkin-Elmer Corp.). Amplification was performed as follows: 30 cycles
at 95°C for 1 min, 55°C for 1 min, and 1 min at 72°C.
Determination of specific activities of 
-galactosidase.
Assays for -galactosidase were performed as described by Miller
(25). Specific activity is given as micromoles of
ortho-nitrophenol (ONP) formed per minute per milligram of
protein, with a molar extinction coefficient for ONP of 0.045 at 420 nm
(25). Protein concentration was determined according to the
method of Lowry et al. (20).
pGh9:ISS1 transposon mutagenesis and selection for
purine auxotrophs.
A pool of pGh9:ISS1 (21)
transposon mutants of L. lactis MG1363 was obtained as
described by Maguin et al. (21), except that GSA medium
containing hypoxanthine (10 µg/ml) and uracil (10 µg/ml) was used.
After resuspension of the mutant library in GSA medium supplemented
with erythromycin but without purine or pyrimidine addition, the cells
were grown at 37°C for 30 min to stop the growth of purine or
pyrimidine auxotrophs. Then, the culture was diluted 100-fold in the
same medium, reaching an optical density at 450 nm of 0.08, and grown
for an additional 1 h. Ampicillin counterselection for auxotrophs
was performed overnight at 37°C after the addition of ampicillin (100 µg/ml) to the diluted culture. After washing and resuspension of the
cells in 0.9% NaCl solution, 100-µl culture aliquots, both undiluted
and 10-fold diluted, were plated on GSA medium containing hypoxanthine
and uracil. After incubation at 37°C overnight, colonies were tested
for purine auxotrophy on GSA medium with and without hypoxanthine.
Cloning and nucleotide sequencing of
purR::pGh9:ISS1 sequences.
A
standard plasmid rescue procedure was used to isolate genomic DNA
adjacent to the transposon insertion point (40). Chromosomal DNA was extracted (1) from MK136 and digested with
EcoRI. Following self-ligation of a diluted sample of the
digested DNA and transformation of strain MT102, transformant MK163 was
isolated. Plasmid pMK1000 isolated from MK163 contained a 1.1-kbp
insert of Lactococcus DNA. Likewise, HindIII
digestion of MK136 followed by ligation and transformation of JM83
resulted in plasmid pJM1000. The inserts of the plasmids were sequenced
with the Thermosequenase kit as recommended by the manufacturer, and
their lactococcal origin was verified by Southern blotting.
Isolation of temperature-resistant
purR::ISS1 recombinants of
purR::pGh9:ISS1 mutants.
Since pGh9:ISS1 mutagenesis creates a duplication of the
ISS1 sequence, the total pGh9:ISS1 plasmid can be
excised by homologous recombination. MK136 was purified to
single-colony isolates on GM17 medium (36) and incubated at
28°C, the permissive temperature for plasmid replication, whereby all
cells with integrated plasmids were counterselected. Single colonies
were then repurified on GM17 medium and incubated at 37°C, the
nonpermissive temperature for plasmid replication, so that without
selection for erythromycin resistance, the plasmids would be lost. More
than 80% of all colonies appearing were found to be
erythromycin-sensitive, temperature-resistant recombinants.
Construction of an insertion mutant.
Competent L. lactis MG1363 cells were transformed with E. coli
plasmid pJM1010, which is unable to replicate in L. lactis. The plasmid contains a selectable Emr marker and an
internal fragment of the purR gene from pMK1000 (see
Results). Transformants with plasmid pJM1010 integrated into the
chromosome were selected and purified on plates containing 1 µg of
erythromycin per ml. The correct insertion mutations in several
transformants were verified by PCR analysis.
RNA extraction.
Strains MK219 (MK177 harboring plasmid
pMK1033) and MK221 (MG1363 harboring plasmid pMK1033) were grown
exponentially in GSA medium supplemented with erythromycin and
guanosine alone (GSA + GR) in one experiment and with the
subsequent addition of adenine plus hypoxanthine (GSA + GR + A + Hx) in another. Erythromycin at 2 µg/ml,
guanosine at 30 µg/ml, hypoxanthine at 15 µg/ml, and adenine at 15 µg/ml were added. At an optical density at 450 nm of 0.6, RNA was
extracted from 5 ml of culture as described previously (18).
Primer extension mapping.
About 10 pmol of oligonucleotide
primer (MKP55) was used in a primer extension experiment as described
previously (18); 10 µg of RNA was used in each reaction.
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RESULTS |
Isolation of purine auxotrophic mutants after ISS1
transposon mutagenesis of MG1363.
From a library of approximately
4,500 pGh9:ISS1 transposon mutants, we obtained 8 purine
auxotrophic mutants by ampicillin counterselection. Using
plasmid rescue, we cloned the DNA regions flanking pGh9:ISS1
from these mutants and determined the nucleotide sequence of the
Lactococcus chromosomal DNA. Putative open reading frames
identified in MK134, MK138, and MK133 showed extensive similarity to
the last part of purK, the middle of purD, and
the last part of purD from L. lactis
(26) and B. subtilis (7) (data not
shown). Interestingly, the product of an open reading frame from MK136
showed extensive similarity to the B. subtilis PurR
repressor (see below).
Nucleotide sequence of the wild-type purR gene and
purR::pGh9:ISS1 fusion junction
from L. lactis.
The nucleotide sequence of the
purR region from L. lactis MG1363 is shown in
Fig. 1. The sequence was determined from
the rescued plasmids pMK1000 and pJM1000, which contain DNA from either site of the pGh9:ISS1 fusion (see Materials and Methods for
details and Fig. 2 for an overview), and
the wild-type sequence of the junction was confirmed by use of PCR
fragments amplified from the purR gene on the MG1363
chromosome with primers MKP72 and MKP90 (Fig. 2). The DNA region
includes an open reading frame of 271 codons marked as purR
in Fig. 1, with a poor putative ribosome binding site (SD2: TGAGA) at
the right distance from the start codon. An alternative ribosome
binding site (SD1: GGAGA), which is closer to the consensus
Shine-Dalgarno sequence, is situated upstream of SD2, but this may be
too far away from the start codon. The reading frame encodes a 30.4-kDa
protein with an isoelectric point of 5.3. Downstream of the
purR gene is a region of dyad symmetry (Fig. 1) which could
serve as a terminator of purR transcription as well as of
transcription in the opposite direction. The downstream region includes
an open reading frame (marked orfP in Fig. 1) reading toward
purR. This reading frame appears to be homologous to those
for signal peptidases from various organisms (data not shown),
including the spsB gene from Staphylococcus
aureus (5). The site of insertion of the
pGh9:ISS1 element was found to be located between the
ribosome binding site and the translational start codon, with the
ISS1 element and the chromosomal nucleotides 92-ATAATAAA-98
duplicated so that they are present on each side of the pGh9 plasmid.
This arrangement creates the sequence
CAGAACCATAATAAATG in front of the
purR reading frame, with the start codon underlined and the
ISS1 sequence in boldface (Fig. 1).
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FIG. 1.
Nucleotide sequence of the purR region from
L. lactis MG1363. The nucleotide sequence of a 1,151-bp
fragment is shown with the translated sequence of two open reading
frames (purR and orfP) aligned with the coding
sequence. Putative ribosome binding sites are underlined and marked by
SD (Shine-Dalgarno). The transcriptional start site is underlined and
marked with +1, and the presence of putative  10 and 35 regions is
likewise indicated by underlining. The location of a PurBox sequence
overlapping the 35 region is indicated by underlining. An inverted
repeat which might function as a  -independent terminator structure
for both purR and orfP transcription is shown by
arrows under the nucleotide sequence. The location of the
ISS1 insertion point in MK177 is shown by double overlining
of the duplicated purR sequence (ATAATAAA).
Primers MKP90, MKP72, and MKP104 are also shown by underlining.
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FIG. 2.
Physical maps of the purR regions in
wild-type and mutant strains. (A) Physical map of the purR
region in L. lactis MG1363. Boxes represent structural
genes, and the transcriptional start site is marked with an arrow. The
putative terminator of purR and orfP
transcription is indicated by T. The purR-derived inserts in
a number of plasmids are shown below the physical map. (B) Integration
point and structure of the pGh9:ISS1 integrative plasmid
after replicative insertion into the chromosome. During the insertion
event, the ISS1 element is duplicated. (C) Physical map of
the purR region in strain JM1010. The shaded regions
indicate duplicated DNA.
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The purR gene from L. lactis is homologous
to the purR gene from B. subtilis.
Figure
3A shows a comparison between the
purR gene products from L. lactis and B. subtilis. The homology of the two genes appears to span the entire
open reading frames, including a PRPP binding motif previously
identified for B. subtilis PurR (39). The two
proteins show 80% overall similarity, with 51% identical amino acids.
When the nucleotide sequence of the purR gene from B. subtilis was reported, it was noted that the encoded protein showed homology in certain regions with purine
phosphoribosyltransferases (39), and later Christiansen et
al. (4) noted a high level of similarity between PurR and
the xanthine phosphoribosyltransferase from B. subtilis.
Figure 3B shows an outline of the PurR protein in which the areas with
homology to phosphoribosyltransferases (exemplified by the adenine
phosphoribosyltransferase from B. subtilis
[38]) are shaded. By homology to the helix-turn-helix domains of LysR family transcriptional activators (33) a
possible DNA binding region was suggested during computer-aided
similarity searches (data not shown). The position of this motif is
also shown in Fig. 3A and B. It should be noted that the homology to the LysR family is restricted to the helix-turn-helix DNA binding domain.
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FIG. 3.
Comparison of PurR from L. lactis with
homologous proteins. (A) Alignment of PurR from L. lactis
with PurR from B. subtilis. A PRPP binding motif is
underlined. Also, a region fulfilling the consensus requirements for a
LysR family DNA binding motif (see text) is underlined. A region with
similarity to a flexible loop found in the three-dimensional structure
of the orotate phosphoribosyltransferase (OPRTase) of Salmonella
typhimurium is also underlined. Asterisks and dots indicate
identical amino acids and semiconservative substitutions, respectively.
(B) Comparison of PurR with phosphoribosyltransferases exemplified by
the adenine phosphoribosyltransferase (APRTase) from B. subtilis. The shaded regions show the extent of similarity between
the two proteins. The locations of the putative LysR family DNA binding
motif and the PRPP binding motif are indicated by black boxes. Also
shown by a black box is a region with similarity to a flexible loop
found in the three-dimensional structure of the OPRTase from S. typhimurium. The loop was inferred to make contact with PRPP. (C)
Comparison of the PurR region from amino acids D130 to L169 with the
homologous regions of the OPRTases from Sulfolobus
solfataricus and Lactobacillus plantarum (accession no.
g2065443 and e199390, respectively) and the APRTases from B. subtilis and Saccharomyces cerevisiae (accession no.
U86377 and z46659, respectively). The flexible loop consensus sequence
(also shown in panel B) was reported previously (28). A
consensus amino acid sequence for this region is shown in the bottom
row.
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The purR gene encodes a transcriptional activator of
purine gene expression.
It came as a surprise to discover that a
transposon insertion in a gene homologous to a repressor gene
would lead to a purine auxotrophic phenotype. The trivial
explanation, that the insertion prevents transcription of a
downstream purine gene, could be rejected, since no sequences similar
to known pur genes were found downstream of purR.
The only open reading frame found downstream of the purR gene is transcribed in the opposite direction and shows high similarity to the SpsB peptidase gene from S. aureus. We were therefore
left with two different explanations. Either PurR is a transcriptional activator and the pGh9:ISS1 insertion abolishes the
translation of the gene or PurR is a repressor and the insertion of the
transposon leads to elevated levels of the repressor protein, with
transcription and the ribosome binding site originating in the
ISS1 element. The elevated levels of the PurR protein could
then shift the binding equilibrium, resulting in constitutive
repression of the purine biosynthetic genes. To test these
possibilities, we transformed parental strain MG1363 with the
multiple-copy rescue plasmid pMK1000, which contained the same genetic
organization as MK136 but at a higher gene dose. If MK136 were
auxotrophic due to elevated repressor levels, then MG1363 transformed
with pMK1000 should also exhibit purine auxotrophy, since it would
contain even higher levels of the repressor. If, however, PurR is a
transcriptional activator, then the presence of an inactivated gene on
the plasmid should not lead to auxotrophy. The presence of pMK1000 in
MK218 was found not to result in a purine requirement when samples were plated on solid GSA medium, in accordance with the first explanation.
To demonstrate that the purine auxotrophy of the
purR::ISS1 mutation was due to the
absence of the PurR protein, a mutant (JM1010) which synthesizes a
truncated PurR protein was constructed. This mutant was constructed by
integration of a plasmid (pJM1010) which carries an internal
purR fragment covering nucleotides 104 to 678 (Fig. 2A) by a
single crossover event, as described in Materials and Methods.
Integrant JM1010 produces a truncated PurR protein lacking the 74 carboxy-terminal amino acids created by the disruption of the
purR gene at position 678 (Fig. 2C). The strain was found to
be purine auxotrophic, demonstrating that an intact PurR protein is
required for growth in the absence of exogenous purines.
As a second test of the positive role of the purR gene in
purine gene expression, we tested the ability of the wild-type
purR gene to complement the
purR::ISS1 mutation in MK136. To do
this, we created an erythromycin-sensitive, temperature-resistant
purR::ISS1 derivative of MK136 in the
following way. The ISS1 element is duplicated during
transposition (Fig. 2A). Therefore, if homologous recombination occurs
between the ISS1 elements, the plasmid will be deleted from
the chromosome, leaving a single ISS1 element in the
purR gene. The resulting strain, MK177, was found to retain the purine auxotrophy of MK136, as judged by its requirement for purines when plated on solid GSA medium.
Subsequently, the intact purR gene was amplified from the
chromosome of MG1363 with primers MKP90 and MKP104 (Fig. 1). The primers were constructed so that a HindIII site was
attached adjacent to position 1 and an XhoI site was
attached adjacent to position 1122 on the sequence map shown in Fig. 1
(Table 3). The PCR-amplified fragment was digested with XhoI
and HindIII restriction enzymes and inserted between the
XhoI and HindIII sites in the E. coli-L. lactis shuttle vector pCI3340 (10). Plasmid
pMK1030 was found to complement the mutation in MK177, since MK216
(MK177/pMK1930) could grow in the absence of purines while MK177
required a purine source, such as hypoxanthine, for growth.
To see how the purR::ISS1 mutation
affects the regulation of purine genes, we transformed MK177 with a
purD-lacLM fusion plasmid, pLN95 (26), and
measured the production of -galactosidase. Table
4 shows the levels of -galactosidase
produced from the plasmid in the wild-type background (SH1) and in the
purR mutant (MK191). The strains were grown in GSA + GR
and GSA + GR + A + Hx. The purR mutation
resulted in a low level of expression of the purD gene
(Table 4), in accordance with the purine auxotrophic phenotype of
MK177. Guanosine was added to support the growth of MK177 and had been
found to deactivate PurR-regulated promoters to a lesser extent than
other purine sources (data not shown).
Autoregulation of purR transcription and mapping of the
transcriptional start site.
Since plasmid pMK1030, which contains
the purR sequence from position 1 to position 1122 (Fig. 1),
was found to complement the purR::ISS1
mutation, the purR gene must be expressed from the plasmid.
This suggestion strongly indicates the presence of a promoter in front
of purR, between nucleotides 1 and 90 (Fig. 1). We
constructed a fusion between the purR promoter and a
promoterless copy of the lacLM genes from Leuconostoc
mesenteroides in plasmid pAK80 (15). The source of the
purR promoter was a PCR-amplified fragment from MG1363
incorporating a PstI site downstream and a
HindIII site upstream of the promoter. In Table 4 we
present data on the production of -galactosidase from the
resulting plasmid, pMK1033, when present in MG1363 or in MK177.
The transformants were grown in GSA + GR (derepressing conditions;
see above) or in GSA + GR + A + Hx (repressing
conditions; see above). The fact that transcription from the promoter
was elevated in the purR background (compare MK219 to
wild-type MK221) suggests that PurR does repress its own synthesis. It
is interesting that both repressing and derepressing conditions
resulted in the same level of transcription from the purR
promoter in the MG1363 background (MK221). This finding could indicate
that purR is autoregulated independently of the effector
molecule. In other words, PurR appears to bind to the purR
promoter under all growth conditions. For unknown reasons, the
derepressed level of transcription in the purR background (MK219) was
reduced by about 50% by the addition of adenine and hypoxanthine.
In order to map the precise location of the promoter, we determined the
5' end of the purR transcript by primer extension analysis
of RNA isolated from MK219 and MK221 using a primer which hybridized to
the beginning of the lacL gene (MKP55). The results are
presented in Fig. 4. This experiment
showed that the first nucleotide to be transcribed (+1) is the adenine
nucleotide at position 75 in Fig. 1. Just upstream of the start site a
consensus 10 sequence (TATAAT) can be identified with a
spacing of 6 nucleotides. Furthermore, spaced by 17 nucleotides a
consensus-like 35 sequence is present (TTGAAT). In
addition, a TGN sequence, which is often found in front of the 10
element in lactococcal promoters, is seen at the correct position. The
amount of extension product was higher in the purR
background (Fig. 4, lane 7) than in the wild-type background (lane 5)
but was independent of purine additions to the growth medium (compare
lanes 5 and 6 and lanes 7 and 8). Although we did not see a reduction
in purR transcript levels in the purR background
upon purine additions, the primer extension experiments confirmed the
data concerning -galactosidase production from fusion plasmid
pMK1033.
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FIG. 4.
Primer extension analysis of the purR
transcriptional start site. An autoradiogram shows primer extension
experiments performed with 10 µg of RNA extracted from MK219 (MG1363
purR-lacLM, lanes 1 and 2) and MK221 (MK177
purR-lacLM, lanes 3 and 4). RNA was extracted from cells
growing exponentially in GSA medium (lanes 1 and 3) or in the same
medium supplemented with purines (lanes 2 and 4). Lanes G, A, T, and C,
sequencing reactions. Asterisks indicate the limits of the nucleotide
sequence, shown on the right, with the transcriptional start site
underlined. The picture was scanned at 400 dpi with a Scan Jet 4c/T
(Hewlett-Packard Co.) and DeskScan II version 2.3 software. The TIF
file was imported into Top Draw version 3.1 for the addition of text.
Lanes 5, 6, 7, and 8 are identical to lanes 1, 2, 3, and 4, respectively, except that the image was acquired with a Packard Instant
Imager. The Instant Imager measures the radioactivity over the surface
of the gel and is more sensitive than autoradiography.
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DISCUSSION |
In the present work, we identified a number of purine
auxotrophic mutants after pGh9:ISS1 mutagenesis of
strain MG1363. Most of the mutants had the transposon
element inserted in genes involved in purine de novo synthesis, but in
one mutant the transposon impaired the expression of a gene for a
putative regulatory protein which showed a high degree of similarity to
the purR gene from B. subtilis. By
complementation of the purR:ISS1 mutant MK177 from L. lactis with plasmid pMK1030 containing the wild-type
L. lactis purR gene, it was verified that the purine
auxotrophic phenotype was the result of the purR mutation.
Does PurR activate purC and purD
transcription through binding to PurBox's?
In the accompanying
report on the regulation of the purC and purD
operons in L. lactis (18), a 13-nucleotide-long
DNA sequence (PurBox; Table 5) which is
present in front of both promoters was found to be important for
high-level expression and purine regulation from the promoters. It was
concluded that the PurBox sequences are binding sites for a
transcriptional activator. In the present study, we showed that PurR
most likely is the transcriptional activator for purC and
purD, since the absence of PurR resulted in decreased
expression of -galactosidase synthesis from the purD
promoter on plasmid pLM95 (26), compared to the expression in the wild-type parental strain MG1363. The purine regulation of the
residual promoter activity was not expected and may indicate that the
mutation present in MK177 does not totally abolish PurR production.
Transcription from the purR promoter was found to be
repressed by PurR, a result which strengthens the inferred bond between
PurR and PurBox's, since the 35 region of the purR promoter overlaps a PurBox (Fig. 1).
The PurBox sequence located at position 76 relative to the
transcriptional start site (18) in both the purC
and the purD promoter regions was shown to be required for
the activation of transcription. The apparent constraint on the
location of the PurBox suggests that PurR and the initiating RNA
polymerase have to form a specific complex involving protein-protein
interactions between the PurR protein and the RNA polymerase. The
autorepression of purR was found to be identical whether the
cells were grown in the presence or in the absence of purine bases
(Table 4), and only the absence of PurR led to elevated activity. These
data suggest that PurR remains bound to the PurBox under all
conditions. If this is the case for all PurBox sequences, which is a
reasonable assumption, then the activation of transcription at the
purC and purD promoters may involve a
conformational change of PurR from a nonactivating to an activating
conformation which recognizes the RNA polymerase. Such an activation
mechanism is known for members of the LysR family of transcriptional
activators (33).
It is interesting to note that two regions in
phosphoribosyltransferases show a high degree of similarity to PurR
(Fig. 3B and C). One of these is the PRPP binding region previously
noted by Weng et al. (39). Another region, which was found
in a homology search of GenBank (Fig. 3C), was previously shown for
orotate phosphoribosyltransferases to constitute a flexible loop. This loop forms a contact with a PRPP molecule, situated in the PRPP binding
site, through a conserved lysine residue (12, 27, 28),
thereby promoting a conformational change. In PurR, a lysine residue in
a similar context was detected at amino acid position 162 (nucleotide
position 582 in Fig. 1; shown in boldface in Fig. 3C); this finding
could suggest that the movement of a flexible loop is involved in the
conformational change leading to the activation of transcription by
binding to PRPP.
Do PurR from L. lactis and PurR from B. subtilis share common mechanisms of action?
We face an
interesting dilemma when we consider that the PurR proteins from
L. lactis and B. subtilis show high overall
similarity (Fig. 3A). The PurR protein from B. subtilis was
reported to function as a repressor (7, 35, 39) which
represses the pur operon only when purines are present in
the growth medium. The activation of transcription by the PurR protein
from L. lactis, on the other hand, is seen only when purines
are absent from the medium. It has been reported that the DNA binding
ability of the PurR protein from B. subtilis is inhibited by
PRPP (39), since the ability of PurR to bind DNA to the
promoter region from the purine operon was detected only at lower PRPP
concentrations. It is reasonable to suppose that the activity of the
PurR protein from L. lactis is also regulated by the level
of PRPP, since it shows 80% overall similarity with the PurR protein
from B. subtilis and since both protein sequences contain a
consensus PRPP binding domain (14, 39). The addition of
adenine and hypoxanthine to the growth medium of both bacteria resulted
in reduced expression of the purine genes, and in both bacteria this
regulation was dependent upon a PurR homolog. It must be kept in mind,
however, that when one is interpreting levels of enzymes encoded by the
intact purine operon in B. subtilis, the regulation of the
pur operon involves an additional guanine- and
hypoxanthine-responsive attenuation system (6).
How the PRPP concentration in B. subtilis was influenced by
the addition of adenine to the growth medium was suggested for B. subtilis to be the consequence of two separate events
(39). Conversion of adenine to AMP by the adenine
phosphoribosyltransferase requires PRPP, so a massive conversion of
adenine to AMP could lead to PRPP depletion. At the same time, the high
AMP pool levels could lead to elevated levels of ADP, which is known to
be a potent inhibitor of the PRPP synthetase from B. subtilis (2). Therefore, the model for PurR regulation
in B. subtilis suggests that a high adenine concentration
leads to a low PRPP concentration, which allows PurR binding.
Accordingly, a low extracellular concentration of adenine would lead to
a high PRPP concentration, which would inhibit DNA binding of PurR. We
believe that DNA binding of PurR from L. lactis is not
inhibited by PRPP, since the high PRPP level which is present when no
adenine is added results in PurR activation and thus DNA binding.
Moreover, PurR appears to bind to PurBox's both in the presence and in
the absence of exogenous purine bases (Table 4 and Fig. 4, lanes 5 and
6).
In B. subtilis, PurR has been shown to protect a region of
more than 60 bp from DNase I attack upon binding to a poorly defined operator site in front of the pur operon (8, 35).
In the core of the protected region (8) we have detected at
position 75 relative to the transcriptional start site a degenerate
PurBox (Table 5) which could serve as the primary binding site. We have also detected DNA sequences resembling PurBox's in front of two additional PurR-regulated genes from B. subtilis,
purA (22) and purR (39), at
about position 40 relative to the suggested transcriptional start
sites, as for the purR gene in L. lactis (this
work). The purA-specific PurBox is a perfect match for the L. lactis consensus sequence, while the PurBox in the
purR promoter region has a C10-to-T mismatch (Table 5). If
the PurBox consensus sequence also applies to the B. subtilis PurR repressor, then the degenerate PurBox in the
purR gene would lead to weaker autorepression and elevated
levels of PurR in B. subtilis relative to L. lactis, in which autorepression involves a perfect PurBox (Table
5). Actually, Shin et al. (35) have presented data
suggesting that PurR from B. subtilis does recognize the
PurBox sequence. A consensus sequence for PurR binding
(GAAC-N24-25-GTTC) was proposed based on its presence in
the pur, purA, and purR promoter
regions. By site-directed mutagenesis and studies of binding of the
region from the pur operon, Shin et al. (35)
obtained data showing that the GAAC sequence was required for efficient
PurR binding, whereas the GTTC sequence was not. Interestingly, the
GAAC sequence required for PurR binding in B. subtilis is
situated in the core of the PurBox sequence proposed above (from G7 to
C10). The authors showed that mutating the GAAC sequence to GAAT, AAAT,
or ATCG resulted in an increase in the apparent
Kd from 7 nM to 16, 52, or 78 nM, respectively
(35). Additionally, they found that methylation of the G7 on
one strand and the G residue complementary to the C10 on the other
strand resulted in a loss of PurR binding (35).
A unifying evolutionary model for PurR action in L. lactis and B. subtilis.
If the PurR proteins from
L. lactis and B. subtilis are homologous, then
both proteins have evolved from a common PurR ancestor. Whether this
nearest ancestor was more similar to one or the other is at present
impossible to deduce, since we have only two PurR homologs to compare,
but the question can be addressed when more PurR proteins are analyzed.
It is reasonable to assume that the ancestral PurR was an activator
which could activate transcription when bound to a PurBox located at
about 76 bp upstream of the transcriptional start site, since both the
L. lactis purC and purD promoters and the
B. subtilis pur operon promoter share this organization.
There is no reason to doubt that this activation would have been
inhibited by PRPP, as it is for the L. lactis protein.
Whether the ancestral PurR protein, upon PRPP binding, would have
been able to repress the promoter from the same binding site cannot be
answered with our limited data, but this type of PurR repression is
known for the pur operon in B. subtilis.
Somewhere in the evolutionary transition from an activator to a
repressor, there must have been a bifunctional PurR protein, which
could both activate and repress transcription from the same site. A
hypothetical model for this transit PurR protein is shown in Fig.
5. The protection of more than 60 bp by
PurR binding in B. subtilis was suggested to be the result
of the DNA being wrapped around the PurR repressor (35).
Alternatively, it could involve more than one PurR unit, so that
massive protection could be the result of PurR multimerization along
the DNA, leading to sequestering of the 35 region in B. subtilis. This possibility, which is in accord with the PurR
binding stoichiometry of six PurR homodimers per 196 bp of the
pur operon in the absence of PRPP (35), is indicated in Fig. 5. Such a polymerization would be dependent upon a
high PurR concentration, since no additional PurBox's can be found in
the promoter regions. That multimerization and transcription activation
in the transit PurR must have been mutually exclusive phenomena is
almost self-evident, since a PurR multimer blocks access to the 10
region. Thus, multimerization could be favored at low PRPP
concentrations, leading to repression, while RNA polymerase binding
could be favored at high PRPP concentrations, leading to enhanced
transcription of the purine biosynthetic genes (Fig. 5).
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FIG. 5.
Hypothetical bifunctional regulator as an intermediate
in the evolution of PurR. The hypothetical bifunctional regulator is
proposed to bind to a PurBox under all conditions, but it can change
between two conformations, depending on the presence of PRPP. The
conformational changes in the transit PurR upon PRPP binding might
favor polymerization at low PRPP concentrations and RNA polymerase
binding at high PRPP concentrations. The model incorporates data for
PurR binding in both B. subtilis and L. lactis.
In B. subtilis, PurR binding to PurBox DNA is only detected
at low PRPP concentrations and only with an extended footprint from
multiple PurR homodimers bound to the DNA. In L. lactis,
PurR binds to DNA with both high and low PRPP concentrations, but
activation is detected only with high PRPP concentrations. The
bifunctional transit PurR protein could be the intermediary state in
the evolution of an activator-regulated system into a
repressor-regulated system with the same regulatory protein, promoter,
and regulator binding site.
|
|
If the Bacillus PurR regulator has gone through such
a transition, one could envision that the pur
operon was originally under activation control but that the evolving
bifunctional transit PurR allowed the introduction of a 35 region,
which created an activator-independent, repressor-controlled promoter.
We are currently testing whether the PurR protein in B. subtilis can activate transcription from the lactococcal
purC promoter, thus resembling the postulated transit PurR
protein. We are also testing whether the PurR activator from L. lactis forms extended footprints at high protein concentrations and whether it can repress the pur operon promoter when
supplied at elevated levels.
We sincerely appreciate the expert technical assistance of
Kristina Brandborg Jensen and Susan Outzen Jørgensen. We thank Hans
Henrik Saxild for help on the plasmid rescues and many stimulating discussions. We also thank Martin Willemoës for many fruitful discussions and for directing our attention to the orotate
phosphoribosyltransferase flexible loop.
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Abstract
Introduction
