Antimicrobial Agents and Chemotherapy, July 1999, p. 1769-1772, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Activation of the 2'-N-Acetyltransferase
Gene [aac(2')-Ia] in
Providencia stuartii by an Interaction of AarP with the
Promoter Region
David R.
Macinga,2
Michael R.
Paradise,1
Milica M.
Parojcic,1 and
Philip
N.
Rather1,2,*
Research Service, Veterans Affairs Medical
Center,1 and Departments of Medicine and
of Molecular Biology and Microbiology,2 Case
Western Reserve University School of Medicine, Cleveland, Ohio 44106
Received 4 January 1999/Returned for modification 23 March
1999/Accepted 7 May 1999
 |
ABSTRACT |
The aac(2')-Ia gene in
Providencia stuartii encodes a
2'-N-acetyltransferase capable of acetylating both
peptidoglycan and certain aminoglycoside antibiotics. Regulation of the
aac(2')-Ia gene is influenced in a
positive manner by the product of the aarP gene, which
encodes a small transcriptional activator of the AraC (XylS) family. In
this study, we demonstrate the sequence requirements at the
aac(2')-Ia promoter for AarP
binding and activation.
| |
TEXT |
Providencia stuartii is a
gram-negative pathogen that is a leading cause of urinary-tract
infections in patients undergoing chronic catheterization (16, 22,
28, 29). Eradication of P. stuartii infections can be
extremely difficult due to the multiple antibiotic resistances that are
often present in this bacterium (11, 15). An intrinsic,
chromosomal acetyltransferase [AAC(2')-Ia] is present in P. stuartii and confers high-level aminoglycoside resistance when
overexpressed (4, 24, 25, 31). In addition, this enzyme
serves a housekeeping role in the O acetylation of peptidoglycan
(21). Intrinsic chromosomal aminoglycoside
acetyltransferases in other bacteria have been described previously,
and a summary of these enzymes has been reported (26).
A central activator of aac(2')-Ia gene
is AarP, a transcriptional activator similar to the MarA, SoxS, and Rob
proteins (14). The MarA, SoxS, and Rob proteins have been
shown to activate an intrinsic multiple-antibiotic resistance phenotype
(Mar) and a defense regulon against superoxide-generating agents in
Escherichia coli (1, 2, 5, 9, 10, 12, 13, 18-20,
30). In addition to activating the
aac(2')-Ia gene, increased expression of AarP activates a Mar phenotype in both P. stuartii and
E. coli (14). Presumably each of these proteins
is capable of activating a common set of genes due to the extensive
homology in their helix-turn-helix DNA binding motif (9).
Prior to this study, it was not clear whether AarP influenced
expression of aac(2')-Ia by a direct
or indirect mechanism. In this report, the
aac(2')-Ia sequences required for the
in vivo activation by AarP are identified. DNA binding studies using
purified AarP protein suggest that activation of
aac(2')-Ia by AarP is mediated by an
interaction with the aac(2')-Ia
promoter region.
Bacterial strains and growth conditions.
Escherichia
coli DH5
(Gibco/BRL, Gaithersberg, Md.) and XL1-Blue
(Stratagene, La Jolla, Calif.) were used as hosts for plasmids. P. stuartii PR50 has been described previously
(24). Luria-Bertani broth was used for the growth of cells,
and the following antibiotics were used at the indicated concentrations
for selections: ampicillin, 150 µg/ml for E. coli and 300 µg/ml for P. stuartii; chloramphenicol, 100 µg/ml for
P. stuartii.
Upstream aac(2')-Ia sequences
required for activation by AarP.
We previously reported that the
presence of aarP on a multicopy pACYC184 derivative (pSF2)
was capable of 10-fold activation of an
aac(2')-lacZ fusion contained on a
separate compatible plasmid, pR401 (14). To determine the
sequences required for this activation, a series of progressive 5'
deletions of the full-length
aac(2')-Ia promoter were constructed.
As shown in Fig. 1, plasmid pR402
contains a 456-bp XhoI-HindIII
fragment inserted into pQF50 digested with SalI and
HindIII. pR403 contains a 357-bp
XmnI-HindIII fragment cloned into pQF50
digested with SmaI and HindIII. pR404
contains a 292-bp AluI-HindIII fragment
cloned into pQF50 digested with SmaI and
HindIII. To construct pR405, plasmid pBC.KS-aac(2') was first digested with ApaLI followed by Klenow treatment to
create blunt ends. This was followed by digestion with
HindIII, which released a 273-bp fragment that was
cloned into pQF50 digested with SmaI and
HindIII. Plasmid pR406 was constructed in two steps. First the aac(2')-Ia promoter region
from pR401 was cloned into pACYC184 as a
BamHI-HindIII fragment. This plasmid was then
linearized with ApaLI, which cuts at position 
49 in the
aac(2')-Ia promoter region end filled
with Klenow treatment in the presence of deoxynucleoside triphosphates.
Religation then created a 4-bp insertion of CATG at position 47. This
promoter fragment was then recloned into pQF50 as a
BamHI-HindIII fragment to create pR406. Each
of these derivatives was electroporated into PR50, and then either pSF2 or pACYC184 was introduced into each strain. The ability of AarP to
activate each promoter derivative was determined by analysis of
-galactosidase accumulation in sodium dodecyl sulfate
(SDS)-chloroform-treated cells by the method of Miller (17).
Plasmid pR401 was activated 9- to 10-fold in the presence of pSF2 (Fig.
1). Deletion derivatives extending to 233 (pR402), 134 (pR403), or
67 (pR404) relative to the start of transcription still supported a
seven- to ninefold activation by pSF2. However, pR405 containing a
deletion extending to an ApaI site at 49 failed to support
activation by AarP. This ApaI site was centered within a
region of dyad symmetry that represented a potential sequence required
for activation by AarP. To examine this possibility, pR406 containing a
4-base insertion of CATG at 47 was assayed for activation. The
presence of this insertion in the context of the full-length promoter
resulted in a severe reduction in the activation conferred by pSF2,
1.5-fold compared to the 10-fold increase seen in pR401.
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FIG. 1.
Deletion analysis of
aac(2')-Ia sequences required for
AarP-mediated activation. A series of 5' deletion derivatives of the
aac(2')-Ia promoter were constructed
and fused to the lacZ gene in pQF50 (7). P. stuartii PR50 containing each deletion derivative fused to
lacZ was then transformed with a compatible plasmid
containing aarP (pSF2) or with a pACYC184 control. The
ability of each aac(2')-Ia promoter
derivative to support activation by AarP was determined by analysis of
-galactosidase (-gal) accumulation according to the method of
Miller (17). A representative experiment is shown, with the
values in parentheses representing the fold induction conferred by the
presence of pSF2 containing the aarP gene, relative to
pACYC184. Standard deviations are also given, and repeat experiments
gave similar induction values to those shown.
|
|
Purification of AarP.
To determine if the observed activation
by AarP was mediated by a direct interaction with the
aac(2')-Ia promoter region, a
His6-AarP protein was constructed by PCR amplification with primers to introduce the His6 tag at the amino terminus.
DNA sequence analysis confirmed that no mutations were introduced from
the PCR amplification. The His6-AarP fusion protein was
shown to function in vivo as indicated by the 48-fold activation of an
aac(2')-lacZ fusion observed in
P. stuartii when His6-AarP was introduced (data not shown). To express the fusion protein via the T7 RNA polymerase, an
EcoRI-SalI fragment containing the
His6-aarP construct and the ribosomal binding
site from pDS56/RBSII (2a) was cloned into pET24(+)
(Novagen) digested with the same enzymes to create pET24.His(6)-aarP.
The His6-AarP protein was purified by affinity
chromatography using Talon resin (Clontech, Palo Alto, Calif.). Initial
attempts to overexpress and purify AarP from E. coli BL21
(DE3) gave reproducibly poor yields. Examination of codon usage for
AarP revealed a number of rare codons for E. coli;
therefore, purification of AarP was subsequently done in P. stuartii PR50 harboring pET24.His(6)-aarP. To introduce the T7 RNA
polymerase gene into P. stuartii, a 4.4-kb BamHI
fragment from pAR1219 (6) containing both gene I from phage
T7 driven by the lac UV5 promoter and the E. coli
lacI gene was cloned into pACYC184 (3) linearized with
BamHI to create p184.T7. Cells were grown at 37°C in 400 ml of Luria-Bertani broth to an optical density at 600 nm of ~0.6.
IPTG (isopropyl--D-thiogalactopyranoside) was added to a
final concentration of 2 mM and cultures were returned to 37°C for an
additional 3 h. Cells were disrupted by passing twice through a
French pressure cell at 10,000 lb/in2, and cellular debris
was removed by centrifuging twice at 35,000 × g for 30 min at 4°C. The His6-AarP protein was purified with Talon
affinity resin by a batch purification method as described by the
manufacturer (Clontech). Protein concentration was determined by the
method of Bradford. The purified His6-AarP fusion protein was visualized on SDS-12% polyacrylamide gels after Coomassie blue
staining and was greater than 90% pure as shown in Fig.
2.
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FIG. 2.
Purification of AarP protein. A His6-AarP
fusion protein was purified from an induced culture of P. stuartii PR50 as described in Materials and Methods. The purity of
the final preparation was examined on an SDS-12% polyacrylamide gel
stained with Coomassie blue. Prestained molecular mass standards
(Bio-Rad) are in lane M, and the estimated molecular masses are
indicated.
|
|
Binding of AarP to the
aac(2')-Ia promoter.
Mobility
gel shift analysis (8) was performed with the purified
His6-AarP protein and fragments of the
aac(2')-Ia promoter region generated
by PCR amplification and end labeled with
digoxigenin-11-2'-deoxy-uridine-5'-triphosphate followed by gel purification.
DNA binding reactions contained 50 mM KCl, 10 mM Tris (pH 8.0), 2 mM
dithiothreitol, bovine serum albumin (0.4 mg/ml), 10% (vol/vol)
glycerol, and 4 fmol of labeled DNA fragment and purified His6-AarP protein as described in the text. Binding
reaction mixtures were incubated for 20 min at room temperature and
then loaded onto 5% polyacrylamide gels. Gels were electrophoresed at
200 V, washed twice for 20 min in 1× Tris-borate-EDTA buffer, and transferred to nylon membranes with a Bio-Rad (Hercules, Calif.) Semi-Dry blotting system. Filters were developed with LumiPhos 530 (Boehringer Mannheim, Indianapolis, Ind.) and exposed to X-ray film.
The ability of AarP to bind the
aac(2')-Ia promoter is demonstrated by
the presence of a DNA fragment with decreased mobility in the presence
of AarP (Fig. 3) relative to that
observed when no AarP protein is present (Fig. 3). In the presence of a
DNA fragment containing the 4-base insertion centered at position 47,
there was no observable binding by AarP, a result in agreement with the
in vivo analysis (Fig. 1). Control experiments using an excess of
unlabeled specific and nonspecific competitor DNAs indicated that the
observed binding was specific for the
aac(2')-Ia promoter (Fig. 3).
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FIG. 3.
Binding of purified His6-AarP to
aac(2')-Ia promoter sequences. Binding
reactions were set up, subjected to electrophoresis on polyacrylamide
gels, and visualized as described in the text. Approximately 2.5 fmol
of labeled wild-type aac(2')-Ia
promoter fragment [Paac(2')] or the mutant promoter with
a 4-bp insertion at 47 (PApaLI) was included in the
reaction mixtures. Purified His6-AarP (20 pmol) and
unlabeled competitor DNA fragments (1.25 pmol) were added to the
binding reactions where indicated. The designation F denotes free
unbound fragment, and C1 indicates an AarP complex with the
aac(2')-Ia promoter.
|
|
Concluding remarks.
The results of this study indicate that
the AarP protein directly interacts with the
aac(2')-Ia promoter region to mediate transcriptional activation. The in vivo activation of deletion derivatives of the aac(2')-Ia promoter
and a 4-bp insertional mutation have indicated that bases at position
47 are important for activation. The purified AarP protein has been
shown to bind the wild-type aac(2')-Ia
promoter region but was unable to bind a mutant derivative containing a
4-bp insertion at position 47. The sequences at position 47 include
a GCA motif centered at position 47, which may be equivalent to the
GCAY motif proposed to be important in SoxS binding (13a).
The AarP protein is a member of the AraC (XylS) family (23)
and like the SoxS and MarA proteins is missing the portion involved in
ligand binding (5, 9, 23, 30). Therefore, like that of SoxS
and MarA, the regulation of gene expression mediated by AarP appears to
be the result of changes in expression (5, 9, 23, 30).
Studies will now focus on the mechanisms that regulate aarP
expression and result in the subsequent activation of
aac(2')-Ia and genes involved in the
multiple-antibiotic resistance phenotype in P. stuartii.
| |
ACKNOWLEDGMENTS |
We are grateful to S. F. J. LeGrice for plasmid pDS56.
This work was supported by a Merit Review award from the Department of
Veterans Affairs.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases BRB-10, Case Western Reserve University, 10900 Euclid Ave., Cleveland, Ohio 44106. Phone: (216) 368-0733. Fax: (216) 368-2034. E-mail: pxr17{at}po.cwru.edu.
| |
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Antimicrobial Agents and Chemotherapy, July 1999, p. 1769-1772, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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