Journal of Bacteriology, July 2000, p. 3874-3876, Vol. 182, No. 13
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Phosphorylated PmrA Interacts with the Promoter
Region of ugd in Salmonella enterica
Serovar Typhimurium
Andrés
Aguirre,
Sergio
Lejona,
Eleonora García
Véscovi, and
Fernando C.
Soncini*
Departamento de Microbiología,
Facultad de Ciencias Bioquímicas y Farmacéuticas,
Universidad Nacional de Rosario, Rosario, Argentina
Received 10 January 2000/Accepted 14 April 2000
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ABSTRACT |
The Salmonella PmrA-PmrB system controls the expression
of genes necessary for polymyxin B resistance. Four loci were
previously identified as part of the regulon, and interaction of PmrA
with the promoter region of three of them was observed. Here we
characterized the interaction of PmrA with the promoter region of
ugd, previously suggested to be regulated indirectly by
PmrA. Our results indicate that PmrA controls the expression of
ugd by interacting with a specific sequence in the promoter
region of this gene.
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TEXT |
The two-component regulatory system
PmrA-PmrB of Salmonella enterica serovar Typhimurium
controls the expression of genes that mediate resistance to polymyxin B
and other cationic antimicrobial polypeptides (6, 8, 18,
22). This is accomplished by modifications of the
lipopolysaccharide that result in both an increased substitution of the
ester-linked phosphate at position 4' of the lipid A with
4-amino-4-deoxy-L-arabinose (4-ARA) and also larger
amounts of 2-aminoethanol esterifying the diphosphates of the core
oligosaccharide (11), decreasing the binding of the
antimicrobial compounds (11, 19, 20). Four different loci
have been identified to be under control of the PmrA-PmrB system: the
ugd (22) and pmrG genes (7)
and the pmrCAB (22) and pmrF operons
(7). ugd, also known as pmrE
(7), was previously identified as pagA
(17). It encodes a putative UDP-glucose dehydrogenase homologous to the products of Streptococcus pneumoniae cap3A
(3) and Streptococcus pyogenes hasB
(4) genes, which is responsible for the conversion of
UDP-glucose into UDP-glucuronate. The pmrG gene is
homologous to the ais gene of Escherichia coli
(7), whose expression is aluminum induced. The
pmrCAB operon encodes both the two-component system
PmrA-PmrB (18) and a hydrophobic polypeptide not required
for polymyxin resistance (6, 21, 22). The pmrF
operon encodes seven polypeptides, some of which have homology to
oxidoreductases, decarboxylases, and aminotransferases, that are
predicted to be part of the UDP-glucuronate
4-ARA pathway (1,
7). Previous work using lacZ fusions strongly
indicated that the regulation of the four PmrA-PmrB-dependent loci was
direct (7, 22). Recently, it was demonstrated that the
expression of pmrCAB, pmrF, and pmrG
is directly controlled by PmrA, while indirect regulation of
ugd by the response regulator was suggested (24).
Here, we characterized the interaction of PmrA with the promoter region
of ugd. We determined that PmrA recognizes a specific sequence in the promoter region of ugd and that
phosphorylation of the response regulator stimulates the interaction.
Interaction of PmrA with the promoter region of ugd.
To
determine if PmrA interacts with the promoter region of ugd
(Pugd), a 354-bp DNA fragment that encompasses and extends 204 bp upstream from the transcriptional start site (24) was amplified by PCR using the PROM UGD-F (5'-CTGAATTCAGGCGCAGCGTG-3') and the PROM UGD-R (5'-AACCCGTCCCGGATATCGTG-3')
oligonucleotides as primers. For DNA shift mobility assays, we
constructed a pmrA His tag fusion gene. This fusion gene was
generated by PCR using primers PmrA-NTF
(5'-GAGGATCCATATGAAGATACTGATTG-3') and PmrA-H6-CTR (5'-TCCAAGCTTAGTGGT GGTGGTGGTGGTGGCTTTCCTCAGTGGCAACC-3') and
then cloned between the NdeI and HindIII
sites of plasmid pT7-7 to obtain plasmid pPB1022. The His-tagged PmrA
protein was purified using a Ni2+-nitrilotriacetic
acid-agarose affinity chromatography column according to the
QIAexpressionist purification protocol (Qiagen) and exhaustively
dialyzed against 20 mM Tris-HCl (pH 7.9)-50 mM KCl. The protein
concentration was determined by the bicinchoninic acid assay (Bio-Rad),
and the protein profile of the purified PmrA-H6 protein was analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The gel mobility assays were performed essentially as described
elsewhere (12) by incubating 40 to 180 pmol of purified
PmrA-H6 with 1 ng of the 354-bp ugd fragment in 1× Sp1
binding buffer without NP-40 (12) in a 40-µl assay. We
detected a shift of the DNA fragment when we used 90 pmol of unphosphorylated PmrA-H6 (Fig. 1). The
interaction was specific, as demonstrated by the efficient competition
by unlabeled homologous DNA. On the other hand, addition of nonspecific
competitor (up to 2.5 µg of salmon sperm DNA) did not affect the
shift. This result demonstrates that PmrA interacts directly with the
promoter region of ugd.
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FIG. 1.
PmrA interacts with the promoter region of
ugd. The electrophoretic shift mobility assay was performed
using the 32P-, 3'-end-labeled 354-bp PCR fragment of the
promoter region of ugd, incubated with purified PmrA-H6, in
the absence or presence of different amounts of either salmon sperm DNA
as a nonspecific competitor (nonsp. DNA) or the unlabeled promoter
region of ugd (unlab. Pugd).
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The PmrA-H6 concentration necessary to induce a shift in our experiment
was comparable to the maximal concentration reported previously
(24). Indeed, when we used DNA fragments from the pmrCAB and pmrF promoter regions, a shift was
observed at PmrA-H6 concentrations similar to those required for the
shift of the ugd promoter fragment (our unpublished
observations). This finding is consistent with previous in vivo
observations that showed that the PmrA-dependent expression of
ugd did not require a different level of activation of the
PmrA-PmrB regulatory system (7, 22). The larger amounts of
PmrA-H6 required to induce a shift under our experimental conditions
could be explained by the 1 mM dithiothreitol (DTT) that was included
in the binding buffer. Reducing agents such as DTT and
-mercaptoethanol have been demonstrated to affect the oligomeric
status of PmrA, reducing its binding capabilities (24) (see
below). Furthermore, while we used a 354-bp fragment that included 326 bp upstream and 25 bp downstream from the initiation codon of
ugd, Wösten and Groisman used a 268-bp fragment that
included 203 bp upstream and 54 bp downstream from the ugd
ATG start codon (24). This suggests that the additional 123-bp sequence used in our assay may play a role in the protein-DNA interaction.
Determination of the PmrA-binding site.
Since the
ugd promoter region did not contain the imperfect inverted
repeat previously described as the PmrA binding site (24),
we decided to determine in this promoter the DNA sequence recognized by
PmrA. DNA-footprinting analysis was performed on both the coding and
noncoding strands. Protein-DNA complexes were allowed to form for 20 min at 25°C in a solution containing 40 mM Tris-HCl (pH 7.5), 0.1 mM
EDTA, 10 mM MgCl2, 50 mM KCl, 0.5 mM phenylmethylsulfonyl
fluoride, 30 µg of bovine serum albumin/ml, 4 µg of salmon sperm
DNA/ml, 20% glycerol, and 1 mM DTT (FTB buffer) in a total volume of
40 µl, after which DNase I digestion and DNA purification were
performed as described previously (2). Using the purified
PmrA-H6 protein, we observed a protected sequence from 
18 to 44,
and from 16 to 44, relative to the transcriptional start site in
the positive and negative strands, respectively (see Fig. 3, below;
other data not shown). Because it has been demonstrated that addition
of reducing agents like DTT or -mercaptoethanol favor the
dissociation of a dimeric form of PmrA (24), the
footprinting analysis was carried out in the absence of DTT. We
observed that protection of the same region was detected with 24 pmol
of PmrA, 1/10 of the amount of the response regulator needed in the
presence of the reducing agent, corroborating the result reported
previously (24). The footprinting analysis findings are
consistent with the large amounts of PmrA that were required to observe
a protein-DNA interaction in the shift assay, since DTT was included in
the binding buffer.
The two-component paradigm supports the concept that phosphorylation of
a transcriptional regulator affects its DNA binding properties
(23). In order to obtain phosphorylated PmrA, we isolated a
truncated form of PmrB fused to a His tag (H6-PmrBc). This form
encompasses the cytoplasmic region of PmrB and retains the autokinase
and phosphotransfer activities (Fig. 2).
pmrBc was amplified by PCR using the PmrB-NTF
(5'-GAGGATCCATATGCGTTTTCAGCGAAG-3') and PmrB-NTR
(5'-AAGGCCTTACCGCCTGGTAACA-3') oligonucleotides and cloned
between the BamHI and HindIII sites of
plasmid pQE32 (Qiagen) to obtain plasmid pPB1023. H6-PmrBc was purified
by following the protocol described above for PmrA-H6. The protein
profile of the purified H6-PmrBc protein was analyzed by SDS-PAGE. The autokinase and phosphotransfer activities of the H6-PmrBc protein were
determined in a 40-µl total volume. One picomole of H6-PmrBc was
incubated in FTB buffer (without the addition of DTT), and 1 mM
[
-32P]ATP (1,850 cpm/pmol; New England Nuclear), in
the absence or presence of 30 pmol of PmrA-H6, for 1, 5, and 20 min at
37°C (Fig. 2). Maximal phosphorylation of PmrA-H6 was observed at 20 min, and it remained stable for at least an additional 20 min (data not
shown).
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FIG. 2.
Autokinase and phosphotransfer activities of H6-PmrBc.
One picomole of the H6-PmrBc protein was incubated with 1 mM
[-32P]ATP in the presence of 30 pmol of PmrA-H6 in FTB
buffer (without DTT) for 1, 5, and 20 min at 37°C. For the negative
control (), H6-PmrB was incubated under the same conditions for 20 min without the addition of PmrA-H6. The reactions were stopped by the
addition of 0.5% SDS. The samples were loaded in an SDS-12% PAGE
gel, transferred to nitrocellulose, and analyzed by autoradiography.
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To evaluate the effect of the phosphorylation of PmrA-H6 on its
interaction with the promoter region of ugd, the
footprinting assay was performed with H6-PmrBc and ATP. PmrA-H6 was
incubated in FTB buffer at 37°C with 1 pmol of H6-PmrBc, in the
presence or absence of 1 mM ATP. The reaction was allowed to proceed
for 20 min, after which the footprinting assay was performed with the
addition of the 32P-labeled ugd fragment.
Phosphorylation of the PmrA-H6 protein resulted in a 10-fold increase
in protection (Fig. 3). This finding indicates that phosphorylation of PmrA enhances its affinity for the
sequence, as has been demonstrated for other related response regulators (10, 13, 14). Analysis of the protected region showed the presence of a direct repeat
(5'-CTTAAT-N5-CTTAAT-3').
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FIG. 3.
Enhanced affinity of phosphorylated PmrA for the
promoter region of ugd. The footprinting assay was performed
on the coding strand of the promoter region of ugd,
incubated with 0, 0.5, 1, 2.5, 5, 10, 15, 30, and 60 pmol of PmrA-H6
and 1 pmol of H6-PmrBc, in the absence or presence of 1 mM ATP and
without addition of DTT. The solid line represents the PmrA-protected
region.
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To determine the extent of phosphorylation of PmrA under the conditions
used for the footprinting assays, aliquots from a parallel assay, in
which 2.5 µl of [-32P]ATP (3,000 Ci/mmol) was added,
were analyzed by SDS-PAGE. The PmrA band was cut from the gel and the
incorporation of 32P was determined using a Wallac 1209 Rackbetta liquid scintillation counter. From three independent
experiments, we determined a concentration of 0.09 ± 0.02 mol of
phosphate per mol of PmrA, indicating that only 7 to 11% of PmrA was
phosphorylated under the conditions used for the footprinting assays.
This result suggests that phospho-PmrA has an increase in affinity much
higher than that calculated from the footprinting assay described
above. (Additional protected areas appeared when large amounts of
phospho-PmrA-H6 were used [Fig. 3]. We are currently analyzing the
role of these protected areas in the PmrA-dependent regulation of the
expression of ugd, using deletions and different DNA point
mutations.)
PmrA is a member of the OmpR family of response regulators in which the
DNA binding motif is a winged helix-turn-helix (15, 16), and
members of this family are known for binding direct repeats (5, 9,
14). Since a similar sequence is also present in the protected
promoter regions of the pmrCAB and pmrF operons (Fig. 4), we propose that the direct
repeat (YTTAAK) is the target site for the PmrA-controlled
expression of this regulon.
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FIG. 4.
Alignment of the promoter regions of ugd,
pmrCAB, and the pmrF operon. The sequences from
the ugd, pmrCAB, and pmrF promoter
regions were piled up from the transcriptional start site. The YTTAAK
direct repeats are boxed, and the proposed 10 regions are marked in
bold. The arrows indicate the transcription start sites identified
previously (24).
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ACKNOWLEDGMENTS |
We thank Esteban Serra for comments on the manuscript.
This work was supported by grants from CONICET (Proyecto PIP 0849) and
from ANPCyT (Proyecto 01-0000-00409) to F.C.S. and a grant from the
Third World Academy of Sciences (Trieste, Italy) to E.G.V. E.G.V.
is a career investigator of the National Research Council (CONICET,
Argentina), and S.L. is a fellow of the same institution. F.C.S. is a
member of the Rosario National University Research Council (CIUNR) and
CONICET and is also an International Research Scholar of the Howard
Hughes Medical Institute.
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FOOTNOTES |
*
Corresponding author. Mailing address: Facultad de
Ciencias Bioquímicas y Farmacéuticas (UNR), Departamento
de Microbiología, Suipacha 531, (2000) Rosario, Argentina.
Phone: 54-341-4370008. Fax: 54-341-4804598. E-mail:
pat-bact{at}citynet.net.ar.
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Journal of Bacteriology, July 2000, p. 3874-3876, Vol. 182, No. 13
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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