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Journal of Bacteriology, August 1998, p. 4219-4226, Vol. 180, No. 16
Department of Biology, McMaster University,
Hamilton, Ontario, Canada L8S 4K1
Received 10 September 1997/Accepted 1 June 1998
Rhizobium meliloti mutants defective in the
phoCDET-encoded phosphate transport system form root
nodules on alfalfa plants that fail to fix nitrogen
(Fix Because phosphate readily forms
insoluble mineral phosphate complexes with other ions found in soil,
the concentrations of soluble or available phosphate detected in soil
solutions are low and generally range from 0.1 to 10 µM
(3). While many Rhizobium strains are able to
grow at these low phosphate concentrations (6), phosphate
limitation often reduces dinitrogen fixation of
legume-Rhizobium interactions by reducing both nodule number and mass (18, 26, 28). In addition, phosphate limitation may
directly affect discrete steps during the nodule formation and
infection process, such as excretion of Nod factors (22) and/or attachment to roots (17). Additional bacterial
phenotypes of symbiotic importance which change in response to
phosphate limitation include the biosynthesis and/or alteration of
rhizobial cell surface carbohydrates such as exopolysaccharides II
(45) and cyclic Our interest in phosphate metabolism in R. meliloti arose
through our genetic analysis of the ndvF locus which is
located on the pExo megaplasmid. The ndvF mutants form
nodules on alfalfa that contain few bacteria and fail to fix
N2 (Fix Analysis of class II suppressor mutations revealed that these mapped to
the phoU and phoB regulatory genes and that
disruption of these genes suppressed the Fix Bacterial strains, plasmids, media, and growth conditions.
The strains and plasmids used in this work are listed in Table
1. Transcriptional lacZ
fusions to pit were made by subcloning the 2.1-kb
EcoRI fragments from pTH354 (wild type) and pTH380 (sfx1) which included the recF-orfA intergenic
region into the EcoRI site of the reporter plasmid pMP220
(31) to create plasmids pTH376 and pTH365, respectively.
Transcriptional lacZ fusions to orfA were made by
deleting the region between the SphI site located in the
orfA gene and the SphI site of the pMP220
polylinker of plasmids pTH376 and pTH365 to create plasmids pTH378 and
pTH367, respectively.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Phosphate Assimilation in Rhizobium
(Sinorhizobium) meliloti: Identification of a
pit-Like Gene
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
). We have previously reported that two classes of
second-site mutations can suppress the Fix phenotype of
phoCDET mutants to Fix+. Here we show that
one of these suppressor loci (sfx1) contains two
genes, orfA and pit, which appear to form an
operon transcribed in the order orfA-pit. The Pit protein
is homologous to various phosphate transporters, and we present
evidence that three suppressor mutations arose from a single thymidine
deletion in a hepta-thymidine sequence centered 54 nucleotides
upstream of the orfA transcription start site. This
mutation increased the level of orfA-pit transcription. These data, together with previous biochemical evidence, show that the
orfA-pit genes encode a Pi transport system
that is expressed in wild-type cells grown with excess Pi
but repressed in cells under conditions of Pi limitation.
In phoCDET mutant cells, orfA-pit expression is repressed, but this repression is alleviated by the
second-site suppressor mutations. Suppression increases
orfA-pit expression compensating for the deficiencies in
phosphate assimilation and symbiosis of the phoCDET
mutants.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
-(1,2)-glucans in Rhizobium
meliloti (5) and lipopolysaccharides in Rhizobium
leguminosarum (32).
) (8, 10). Two genetically
distinct classes (I and II) of second-site mutations (sfx)
which suppressed the ndvF Fix phenotype were
identified (25), and an analysis of those mutations together
with a characterization of the ndvF locus has been the subject of several recent reports (1, 2, 37). The
ndvF locus was shown to contain four genes,
phoCDET, that encode an ABC-type high-affinity system
for the uptake of phosphate and possibly phosphonates into the cell
(2, 37). The phoCDET mutants were found to
grow poorly in minimal media containing 2 mM orthophosphate
(Pi) as a sole source of phosphorus (2). Class I
suppressor mutations (sfx1, sfx4, and
sfx5) are tightly linked in transduction, and we previously
reported the localization of the sfx1 locus to an 18-kb
BamHI fragment in the cosmid clone pTH56 (25).
Here we show that sfx1 possesses two genes, orfA and pit, and that the deduced Pit protein is homologous with
phosphate transporters such as the Pit protein of Escherichia
coli.
and
Pi growth phenotype of phoCDET
(ndvF) mutants (1). We showed that while the
phoB gene was required for expression of the
phoCDET genes, phoB appeared to play a
negative role in regulating expression of the orfA-pit
genes. Here we demonstrate that the sfx1 mutation is a
promoter-up mutation which increases expression of the
orfA-pit operon and allows Pi uptake via the
OrfA-Pit system in a phoCDET mutant background. We
conclude that this increased Pi uptake is responsible for
the symbiotic phenotype associated with phoCDET
mutations.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids
-galactosidase assays, the
LBmc-grown cells, supplemented with tetracycline at 2 µg/ml for the
strains containing the pMP220-derived plasmids, were washed once with
MOPS P0 and resuspended in this medium. Five-milliliter volumes of MOPS
P0 and MOPS P2 were inoculated with 20 (OD600, ~0.05) and
5 (OD600 ~0.015) µl of cells, respectively, and the
cells were grown for 38 h before the assay was performed.
DNA manipulation and genetic techniques. Cloning procedures, including DNA isolation, restriction digestions, ligation, and transformation, were performed as described by Sambrook et al. (29). We cloned the wild-type orfA-pit region as a 4.8-kb HindIII-SacI fragment from Rm1021 DNA into plasmid pUC118. One such plasmid, pTH354, was identified via colony hybridization by using a formamide-based hybridization solution at 42°C (described in reference 8) and a digoxigenin-labeled 4.8-kb HindIII-SacI fragment from pTH90 as a probe. Annealed probe was detected with anti-digoxigenin antibody conjugated to alkaline phosphatase (AP).
Conjugal mating with MT616 as the helper strain, Tn5 mutagenesis, and homogenizations (with pPH1JI) were performed as previously described (9, 13, 44).DNA sequencing and sequence analysis.
DNA sequencing of
overlapping deletions was performed on single-stranded DNA by using the
dideoxy chain termination method as described in the U.S. Biochemicals
protocol for the Sequenase 2.0 enzyme and by using
[
-35S]ATP (NEN DuPont). Single-stranded DNA was
obtained from host strain XL-1 Blue (Stratagene) following infection
with the helper phage M13K07 (36). Both DNA strands were
sequenced by using the 
20 (lacZ) primer
(5'-GTAAAACGACGGCCAGT-3') and the IS50 primer (5'-TCACATGGAAGTCAGATCCT-3'). The recF-orfA
intergenic region was amplified via the PCR by using primers 41, 5'-AAGTCGCTCAGTTTCAGGCGTGTGAGA-3', and 55, 5'-CCCATGACGGTGCGCGAATGATCGG-3' (see Fig. 4). Following gel
purification, the sequences of the amplified 359- and 360-bp fragments
were determined on an ABI automated DNA sequencer by using the
above-described primer 41.
AP and -galactosidase assays.
The AP activity was
measured as described by Yarosh et al. (44) except that the
cells were centrifuged before the OD420 was measured. The
AP activity was calculated by using the following formula: (1,000 × OD420)/(OD600 × 
T), with
T being the reaction time (in minutes).
-galactosidase assay was performed, and specific activities in
Miller units were calculated as previously described (1).
Determination of transcriptional start sites.
Total RNA was
isolated from MOPS P2 medium-grown cells by using the procedure
outlined in the RNeasy Midiprep kit (Qiagen). Three different primers
(primers 55, 81, and 82 [see Fig. 4]) were labeled with
[
-33P]ATP (NEN Dupont) and polynucleotide kinase
(NEB). Thirty micrograms of RNA was annealed to labeled primer (4 × 105 to 1.5 × 106 cpm) and extended
with Moloney murine leukemia virus reverse transcriptase for 90 min at
42°C. To align the transcriptional start sites, the same primers were
used in conjunction with template DNA (pTH191, sfx1) in a
standard sequencing reaction by using the Sequenase kit (Amersham,
Oakville, Ontario, Canada).
Transport assays. For transport assays, cells were precultured in LBmc, washed three times with MOPS P0, and subcultured into MOPS P2. Cells grown aerobically for 24 h at 30°C were harvested by centrifugation, washed four times with MOPS P0, and resuspended to an OD600 of 10 in MOPS P0. Cells were diluted 1:20 into MOPS P0 and equilibrated for 5 min at 30°C. Uptake was initiated by the addition of [33P]orthophosphoric acid (NEN DuPont) to a final concentration of 4 µM (175 Ci/mol). Aliquots were removed from the assay at different time points, placed on Millipore HAWP 02500 nitrocellulose filters (0.45-µm pore size; presoaked in 1 M K2HPO4-KH2PO4 [pH 7.0]), and immediately washed with 40 mM MOPS-20 mM KOH-20 mM NH4Cl-100 mM NaCl-1.2 mM CaCl2-2 mM MgSO4. Filters were dried, placed in scintillation liquid (BCS; Amersham), and counted. All transport assays were performed in triplicate.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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sfx1 suppresses the Pi transport and growth
phenotypes of phoCDET mutants.
Subsequent to
the identification of sfx1 as a second-site mutation which
suppressed the Fix symbiotic phenotype of
ndvF mutants (25), we established that the
ndvF locus contains four genes, phoCDET,
which encode a phosphate transport system (2). Since
phoCDET mutants grow slowly in medium containing 2 mM
Pi and are defective in Pi transport
(2), we wished to determine whether sfx1
restored a wild-type growth and phosphate transport phenotype to
phoCDET mutants.
490 mutant, RmG490, grew
slowly relative to the wild type, Rm1021, in medium containing 2 mM Pi, the doubling time for the phoC490
sfx1 strain, RmG762, was similar to that of the wild
type (Fig. 1). Additional
experiments revealed that sfx1 also suppressed the
slow-growth phenotype observed when either the phoCDET
deletion (G439) mutants or the phoT491 insertion mutants were grown in MOPS-2 mM Pi (data not shown).
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), RmG490
(phoC
), RmG762 (phoC
), RmG822 (phoC
), RmG830
(phoC
), and RmH842 (phoC
) in MOPS-buffered minimal
medium supplemented with 2 mM Pi. Each time point represent
the average of triplicate values. The growth curve for strain RmG821
[phoC490 mutation together with a Tn5
insertion in the sfx1 locus
(pit10::Tn5), showed Pi uptake
rates similar to those of the phoC mutant RmG490. These
results established that the sfx1 mutation restored
wild-type phosphate transport and growth phenotypes to the
phoC mutant. To further investigate the genetic nature
of the sfx1 locus, we first delineated the minimal gene region required for sfx1-mediated suppression.
|
), RmG762 (phoCLocalization and nucleotide sequence of sfx1.
The
sfx1 locus was previously located to the 18-kb
BamHI fragment on the pTH56 cosmid (25), and as a
first step in the delineation of sfx1, a 12-kb
HindIII fragment internal to the 18-kb region was
subcloned into pRK7813 to give pTH90. This fragment contained the
entire sfx1 locus since RmG490 (phoC490)
pTH90 transconjugants formed Fix+ nodules on alfalfa and
grew like the wild-type strain in medium containing 2 mM phosphate
(data not shown). Tn5 insertions which mapped within the
12-kb insert of pTH90 were isolated, and seven of these plasmids
were used to generate pTH90 deletion derivatives in which various
amounts of the 12-kb insert region had been removed (Fig.
3, upper diagram). Fragments from the
12-kb region were also subcloned into pRK7813 (Fig. 3). The symbiotic
Fix phenotype and/or phosphate growth phenotype of RmG490
(phoC490) transconjugants carrying the various
plasmids allowed us to deduce that the entire sfx1 locus was
located between the EcoRI1 and
EcoRV1 sites indicated on the map of pTH276 in
Fig. 3.
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490 sfx1) chromosome (Fig. 3),
and the physical structure of the resulting recombinants was confirmed
by Southern blot analysis (data not shown). The 2 and 12
recombinants grew like the suppressor strain RmG762 in MOPS medium
containing 2 mM Pi, whereas the 16, -3, -J, -5, -10, -23, and -10A recombinants grew poorly (data not shown and Fig.
1). In addition, 10 and 23 recombinants formed
Fix nodules when tested on alfalfa plants (data not
shown). These results confirmed that sfx1 was located
between the 2::Tn5 insertion and the
EcoRI3 site. The nucleotide sequence of
this 2,828-bp region was determined.
Analysis of the nucleotide sequence of the sfx1 locus. Analysis of the nucleotide sequence revealed the presence of two complete open reading frames designated orfA and pit (see below) and (Fig. 3). The orfA stop codon (TGA) and the pit start codon (ATG) overlapped at the adenosine, suggesting that both genes are transcribed as a single transcript. The G+C contents of orfA and pit were 62 and 64%, respectively. There was a strong bias for G and C at the third position of the codons (83% for orfA and 87% for pit). This bias together with the codon usage of orfA and pit suggested that these genes were expressed in R. meliloti (24, 38).
A partial open reading frame initiating 234 bp upstream from orfA and whose 176-amino-acid product is homologous to the N-terminal regions of many RecF proteins, including Caulobacter crescentus (27), was detected (Fig. 3). An additional partial open reading frame (designated orf2), initiating 114 bp downstream of pit, was also found. Neither recF nor orf2 was part of the sfx1 locus since recF12::Tn5 and
orf22::Tn5 recombinants of strain RmG762 (phoC490 sfx1) retained the suppressor
phenotype (see above). An ATG start codon preceded by a potential
ribosome binding site was found for each gene (CGGA-N6-ATG for
recF, TGGA-N6-ATG for orfA, GAGA-N7-ATG for
pit, and GAGA-N7-ATG for orf2).
GenBank searches (16) revealed that the 334-amino-acid
R. meliloti Pit protein (RmPit) was similar in sequence
to a large family of prokaryotic and eukaryotic phosphate transport
proteins (42, 43; for a review, see reference
20), including the low-affinity Pit phosphate
transport protein of E. coli (accession no. P37308; EcPitA)
(30) and the phosphate-repressible phosphate permease
of Neurospora crassa (accession no. P15710;
NcPho-4+) (21). In addition, RmPit showed
similarity to numerous uncharacterized proteins, including the HI1604
protein of Haemophilus influenza (accession no. P45268)
(15) and the EcPitB protein of E. coli (accession
no. P43676) (4). Consistent with its homology to
Pi transport proteins, an analysis of the RmPit sequence
employing the TopPred II program (11) revealed nine
"certain" membrane-spanning domains.
GenBank searches revealed that the 214-amino-acid OrfA protein had 21%
amino acid identity with the deduced 226-amino-acid HI1603
protein of H. influenzae (accession no. P44271)
(15). This result was interesting since the
HI1603 gene is located directly upstream (25 bp) from the
pit-like HI1604 gene of H. influenzae (see above). The conserved location of orfA-pit-like genes
suggests that they are functionally related.
The sfx1 mutation contains a single T deletion in a
hepta-T sequence upstream of orfA.
To investigate whether
the sfx1 mutation lay in the orfA-recF intergenic
region, the XhoI/EcoRI3 and
EcoRV1/EcoRI3 fragments from the sfx1 locus (Fig. 3) in plasmids pTH396 and
pTH397, respectively, were recombined via a
Campbell-type single crossover into the RmG490
(phoC490) genome (see Materials and Methods).
The presence of the sfx1 mutation was then determined by
screening the recombinants for suppression of the mucoid colony
phenotype of phoCDET mutants as observed on
low-osmolarity GYM medium (25). Of 70 pTH396 recombinants examined, 12 formed dry colonies and thus suppressed the mucoid phenotype of RmG490. On the other hand, of 70 pTH397 recombinants examined, none showed a suppressor phenotype.
Together, these data indicated that the sfx1 mutation lay in
the orfA-recF intergenic region.
80 to 86
upstream from orfA, was reduced to six thymidines in the
sfx1 mutant sequence (Fig. 4).
A 359-bp fragment containing the complete recF-orfA
intergenic region was PCR amplified from the wild type, Rm1021, and the
two other class I suppressor strains, RmG203 (sfx4) and
RmG204 (sfx5) (25). The nucleotide sequences of
the amplified fragments revealed that all three were identical except
that sfx4 and sfx5 mutant sequences, like the
sfx1 sequence, contained six thymidine residues in place of
the seven thymidines present in the wild-type sequence. Thus, all three
independently isolated class I suppressor mutants (sfx1,
sfx4, and sfx5) carried the same thymidine
deletion mutation. While this finding was surprising, we note that
repeat sequences, such as the run of seven thymidines, are known to be
mutational hot spots (23).
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sfx1 increases orfA and pit
expression.
To investigate whether the sfx1
mutation altered the level of orfA-pit expression, we
constructed transcriptional lacZ fusions to the
orfA and pit genes from both the wild-type and
sfx1 loci in the broad-host-range reporter plasmid pMP220
(see Materials and Methods). The resulting plasmids were transferred
into the wild-type Lac R. meliloti
strain, RmG212, and the -galactosidase (Fig.
5b) and AP (Fig. 5a) activities of the
transconjugants were measured after 38 h of growth in
MOPS-buffered medium containing either no phosphate (MOPS P0) or 2 mM
inorganic phosphate (MOPS P2). High and low AP activities confirmed
that the cells were grown under phosphate-deficient and
phosphate-sufficient conditions, respectively (Fig. 5a, data sets 1, 2, and 4).
|
). Each datum point
represents the average of triplicate values ± standard error
(error bar).
background
(RmH667). In this background, levels of both orfA and
pit expression directed from the wild-type locus were very low regardless of whether the cells were cultured in the presence (2 mM) or absence of inorganic phosphate (Fig. 5b, data sets 3 and 5). On
the other hand, while the expression of sfx1-orfA and sfx1-pit gene fusions was reduced by 23 to 30% in the
phoC mutant relative to that in a wild-type background,
their levels of expression were still two to three times higher than
the levels of the corresponding wild-type fusions in a wild-type
background. A two- to threefold increase in orfA-pit
expression thus appears to be sufficient to allow suppression of the
phoCDET phenotypes. We note that the high AP activity
detected in phoC mutant cells cultured in medium containing 0 or 2 mM Pi suggests that these cells are
starved for Pi (Fig. 5a, data sets 3 and 5) (see also
reference 1).
The modest increase in orfA-pit transcription which resulted
from the sfx1 mutation prompted us to determine whether an
increase in the copy number of the wild-type orfA-pit locus
would be sufficient to allow the phoC mutant to grow in
medium containing 2 mM Pi. The wild-type and sfx1
orfA-pit gene regions were therefore cloned into the
broad-host-range vector pRK7813, and RmG490 (phoC)
transconjugants carrying these plasmids (pTH391 and pTH348,
respectively) grew like the wild-type strain, Rm1021, in MOPS P2
(data not shown). We therefore conclude that the increase in
orfA-pit expression resulting from the increase in the copy
number of the wild-type locus is sufficient to suppress the slow growth
of the phoC mutant in medium containing 2 mM
Pi.
The orfA-pit transcriptional start site lies 29 bp upstream of orfA. To investigate the relationship between the sfx1 mutation and the orfA-pit promoter, we mapped the orfA transcriptional start site by primer extension analysis. mRNA was isolated from strains in which the copy numbers of the sfx1 and wild-type orfA-pit regions were increased via the plasmids pTH391 and pTH348, respectively. A single transcriptional start site, lying 29 bp upstream from orfA, was identified with three separate primers (Fig. 4 and data not shown). The extension product observed from cells carrying additional copies of the wild-type orfA-pit gene region was much less intense than the product from cells carrying additional copies of the sfx1 locus (Fig. 4, lanes 1, 4, and 5). Moreover, in strains carrying only the chromosomal copy of the orfA-pit gene region, a faint extension product was observed with mRNA isolated from sfx1 strains while no product was detected in wild-type cells (Fig. 4, lanes 2 and 3). These results confirmed that the sfx1 mutation increased orfA transcription, and the data also suggest that under growth conditions (2 mM Pi) in which the wild-type orfA-pit operon is maximally expressed, the actual level of transcription is low. These conclusions are consistent with the results from lacZ gene fusion studies described above.
We identified10 and 35 consensus-like hexamers (TAT GAC
and TTTCCC, respectively), followed by the
hepta-thymidine repeat (51 to 57), upstream from the
transcriptional start site. Partially overlapping the
heptathymidine repeat site of the sfx1 mutation, we
identified an 18-bp element which contained 11 bp identical to the
consensus binding site for the E. coli PhoB protein
(Fig. 4).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The data presented in this report and previous biochemical data
(37) suggest that the orfA-pit operon of
R. meliloti encodes a phosphate transport system whose
expression is regulated by the amount of available inorganic
phosphate. The orfA-pit genes are expressed in cells
grown in the presence of excess phosphate (2 mM Pi),
whereas under Pi limiting conditions orfA-pit
transcription is repressed (Fig. 5). Unlike the latter wild-type
pattern of orfA-pit regulation, our data show that in
phoCDET mutant cells, transcription of the
orfA-pit operon is repressed, even in cells cultured with
excess Pi (2 mM) (Fig. 5b, data sets 3 and 5). Thus, phoCDET mutants grow poorly in medium containing 2 mM
Pi and show a reduced Pi transport phenotype
not because they lack the PhoCDET transport system but because
orfA-pit expression is repressed. Hence, the Pi
assimilation phenotypes resulting from phoCDET mutations are eliminated by secondary mutations such as sfx1 which
increase orfA-pit transcription. The fact that
sfx1 was identified as a suppressor of the Fix
phenotype of phoCDET mutants reinforces the suggestion
that the phoCDET Fix phenotype is a direct
consequence of the Pi assimilation phenotype of these
mutants.
In E. coli and Acinetobacter johnsonii, there is physiological evidence that a Pit-like phosphate transport system, which transports Pi as a neutral metal phosphate complex, operates during conditions of Pi excess and that under Pi limiting conditions a separate high-affinity phosphate transport system is employed for Pi uptake (PstSCAB in E. coli) (33-35, 39, 40). While the proposed role for the Pit protein in R. meliloti is similar to that of the E. coli Pit protein, our data clearly suggest that orfA-pit expression is phosphate regulated in R. meliloti whereas the E. coli Pit transport system is believed to be constitutively expressed (40).
Why phoCDET mutations result in repression of
orfA-pit expression is unclear. However, we recently showed
that this repression is mediated through the transcriptional activator
PhoB since mutations which inactivate the phoB gene relieve
this repression (1). In wild-type cells, PhoB also appears
to mediate the phosphate-dependent regulation of orfA-pit
expression, since orfA-pit expression is constitutive in a
phoB mutant background (1). In view of the above
data, it is particularly interesting to find a PhoB-like binding site
in the orfA promoter. In E. coli, genes whose
expression is activated by PhoB contain a PhoB binding site in the 35
region of the promoter. Similarly, the R. meliloti
phoC promoter contains a PhoB-like binding site in the 35
region, and phoC expression is positively regulated by
phoB (2). The PhoB-like binding site of the
R. meliloti orfA-pit wild-type promoter is centered at
62.5, and we suggest that this atypical positioning reflects the
negative regulation of orfA-pit expression by PhoB.
While the single thymidine deletion (sfx1) mutation increases the basal expression and strength of the orfA-pit promoter, expression is still regulated by the level of available phosphate. Indeed, the sfx1 mutation appears to increase the sensitivity of the orfA-pit promoter to regulation by phosphate, since sfx1-directed orfA-pit transcription was Pi regulated in both the wild-type and phoC backgrounds (Fig. 5b, data sets 2 to 5), whereas (as noted above) wild-type orfA-pit showed only very low expression in the phoC background (Fig. 5b, data sets 3 and 5). Given the location of the sfx1 mutation and PhoB-like binding site (Fig. 4), it is not surprising that sfx1 transcription is still subject to phosphate-dependent regulation. Further studies are clearly required to verify the PhoB binding site and to investigate how the deletion mutation increases the strength of the orfA-pit promoter.
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ACKNOWLEDGMENTS |
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This work was supported by operating and strategic grants from the Natural Sciences and Engineering Research Council of Canada to T.M.F.
We thank Nathan Falcioni for technical assistance in identifying the sfx3 and sfx4 mutations, Bob Watson for the yeast extract fraction, and all members of the Finan laboratory for comments and discussions.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, McMaster University, 1280 Main St. West, Hamilton, Ontario, Canada L8S 4K1. Phone: (905) 525-9140, ext. 22932. Fax: (905) 522-6066. E-mail: FINAN{at}MCMASTER.CA.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Bacteriology, August 1998, p. 4219-4226, Vol. 180, No. 16
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