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Antimicrobial Agents and Chemotherapy, August 2001, p. 2238-2244, Vol. 45, No. 8
Department of Microbiology, University of
Guelph, Guelph, Ontario N1G 2W1, Canada
Received 10 November 2000/Returned for modification 19 March
2001/Accepted 5 May 2001
The gene coding for aminoglycoside
2'-N-acetyltransferase Ia [AAC(2')-Ia] from
Providencia stuartii was amplified by PCR and cloned.
The resulting construct, pACKF2, was transferred into Escherichia coli for overexpression of AAC(2')-Ia as a
fusion protein with an N-terminal hexa-His tag. The fusion protein was isolated and purified by affinity chromatography on
Ni2+-nitrilotriacetic acid agarose and gel permeation
chromatography on Superdex 75. Comparison of the specific activity of
this enzyme with that of its enterokinase-digested derivative lacking
the His tag indicated that the presence of the extra N-terminal peptide does not affect activity. The temperature and pH optima for activity of
both forms of the 2'-N-acetyltransferase were 20°C and
pH 6.0, respectively, while the enzymes were most stable at 15°C and
pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2')-Ia at 20°C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2'-amino group and a concentration of acetyl
coenzyme A fixed at 10 times its Km
value of 8.75 µM. Under these conditions, gentamicin was determined
to be the best substrate for the enzyme in terms of both
Km and kcat/Km
values, whereas neomycin was the poorest. Comparison of the kinetic
parameters obtained with the different aminoglycosides indicated that
their hexopyranosyl residues provided the most important binding sites
for AAC(2')-Ia activity, while the enzyme exhibits greater tolerance
further from these sites. No correlation was found between these
kinetic parameters and MICs determined for P. stuartii
PR50 expressing the 2'-N-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.
The gram-negative bacterium
Providencia stuartii and other species of
Providencia, Proteus, and Morganella comprise the
Proteeae, and all members have been shown to O acetylate
peptidoglycan (4). Peptidoglycan is a rigid cell wall
heteropolymer comprised of repeating N-acetylglucosaminyl
and N-acetylmuramyl residues with an attached
tetrapeptide. The O acetylation of peptidoglycan occurs at the C-6
hydroxyl group of muramyl residues, at levels ranging from 20 to 70%
(recently reviewed in reference 5). This modification to
peptidoglycan confers resistance to muramidases and has also been
proposed to modulate the activity of endogenous peptidoglycan hydrolases, including the autolysins (11). Other important
pathogens that modify their peptidoglycan in this manner include
Staphylococcus aureus and Neisseria gonorrhoeae
(11). Little is known about the pathway for the O
acetylation of peptidoglycan, but studies with P. stuartii suggest that a previously identified aminoglycoside acetyltransferase may contribute to the process (9-11).
The aminoglycoside acetyltransferases are comprised of four classes of
enzymes, designated AAC(1), AAC(2'), AAC(3), and AAC(6'), according to
the site of acetylation of the deoxystreptamine core of the
aminoglycoside antibiotic (16). These enzymes are common among both gram-negative and gram-positive bacteria. Most of the genes
encoding these enzymes are plasmid borne, but an exception to this
general rule is the gentamicin 2'-N-acetyltransferase [AAC(2')-Ia] from P. stuartii, which is chromosomally
encoded. In wild-type P. stuartii, the aac(2')-Ia
gene is normally expressed at low levels (15), and it is
regulated in part by a small transcriptional activator, AarP, and at
least two other trans-acting negative regulators (14,
15).
Mutant strains of P. stuartii that either under- or
overexpress AAC(2')-Ia have correspondingly lower or higher levels of peptidoglycan O acetylation, in addition to altered MICs of
aminoglycoside antibiotics (10, 11). These changes to the
levels of O acetylation have been shown to result in altered cell
morphologies. Further in vitro characterization of the enzyme suggested
that it could use peptidoglycan metabolites, in addition to acetyl
coenzyme A (acetyl-CoA), for the acetylation of both aminoglycosides
and peptidoglycan (9). We have recently proposed that
AAC(2')-Ia transfers acetate to peptidoglycan from an integral membrane
protein which serves to translocate it from cytoplasmic pools of
acetyl-CoA to the outer surface of the cytoplasmic membrane
(5).
To provide sufficient quantities of AAC(2')-Ia for detailed enzymatic
studies and delineation of its role in the pathway for peptidoglycan O
acetylation, we have cloned its gene into a T7-based overexpression
vector. The enzyme was purified to homogeneity, and its
Michaelis-Menten parameters with different aminoglycoside substrates and pH and temperature optima were determined. Analysis of
these kinetic parameters provided further support for an additional function of AAC(2')-Ia in P. stuartii besides
conferring aminoglycoside resistance.
Bacterial strains and growth conditions.
The strains and
plasmids used in this study are listed in Table
1. Escherichia coli
DH5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2238-2244.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Overexpression and Characterization of the
Chromosomal Aminoglycoside 2'-N-Acetyltransferase of
Providencia stuartii
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
and E. coli BL21 were maintained either in
Luria-Bertani (LB) broth or on LB agar, which was supplemented with 30 µg of kanamycin sulfate per ml in the case of pET30a(+)-containing strains (Novagen). All cultures were grown at 37°C with aeration, unless otherwise stated.
TABLE 1.
Strains and plasmids used for this study
Enzyme and biochemicals. Tryptone, LB broth and agar, yeast extract, and Bacto agar were purchased from Difco Laboratories (Detroit, Mich.). Tobramycin was purchased from ICN Biomedical (Costa Mesa, Calif.), while all other aminoglycosides, enterokinase, and reagents were purchased from Sigma (St. Louis, Mo.). Gentamicin was supplied as a mixture of gentamicins C1 (<45%), C1a (<35%), and C2 (<30%). Ni2+-nitrilotriacetic acid (Ni2+-NTA) agarose was obtained from Qiagen (Mississauga, Ontario, Canada), and Superdex 75 was from Amersham Pharmacia Biotech (Baie d'Urfé, Quebec, Canada). Restriction enzymes were purchased from New England Biolabs (Mississauga, Ontario, Canada), while all other DNA-modifying enzymes were supplied by Roche Diagnostics (Laval, Quebec, Canada).
Construction of AAC(2')-Ia overexpression plasmid pACKF2.
The aac(2')-Ia gene, originally obtained from pSCH4500
(15) (kindly provided by P. Rather), was subcloned from
pUCP26 (provided by L. Burrows) into pBAD24 (pACKF1). Induction trials
with pACKF1 revealed poor protein expression, and hence an
alternative clone was constructed. Using pACKF1 as a template,
aac(2')-Ia was amplified using primers
5'-GAGGAATTCACCATGGGCATAGAATAC-3'
and 5'-TAGAGGATCGAATTCTGATTACCACTG-3', which introduced unique NcoI and EcoRI
restriction sites (underlined), respectively, to facilitate cloning.
The gene was amplified with Pwo DNA polymerase (Roche) by 30 cycles consisting of 3 min at 94°C, 45 s at 94°C, 30 s at
60°C, 45 s at 72°C, and 7 min at 72°C. The 512-bp fragment
was isolated using the QIAquick Gel Extraction Kit (Qiagen), digested
with NcoI and EcoRI (New England Biolabs) according to the manufacturer's instructions, and then ligated into
plasmid pET30a(+) to generate pACKF2, encoding an N-terminal poly-His-tagged AAC(2')-Ia. Potential clones were initially screened in
transformed E. coli DH5, and then pACKF2 was transferred
into E. coli BL21(
DE3) (AJC105) for subsequent
expression. Confirmation of the presence of the complete
aac(2')-Ia insert in the construct was obtained by DNA
sequencing (Laboratory Services Division, University of Guelph).
Overexpression of AAC(2').
LB broth (500 ml) supplemented
with 30 µg of kanamycin per ml was inoculated with a sample of a 16-h
culture of freshly transformed AJC105 and grown at 37°C in a rotary
shaker at 250 rpm until the optical density at 600 nm reached 0.6. Sterile isopropyl-
-D-thiogalactopyranoside (IPTG) was
added to a final concentration of 1 mM. Cells were allowed to grow for
an additional 2 h prior to harvesting by centrifugation at
5,000 × g for 15 min.
Purification of AAC(2')-Ia. The cell pellet was resuspended in 20 ml of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole) in the presence of complete EDTA-free protease inhibitor cocktail tablets (Roche), 10 µg of RNase per ml, and 5 µg of DNase per ml. Cells were disrupted using a sonicator ultrasonic liquid processor (Heat Systems Inc., Toronto, Ontario, Canada) set at 50% maximal output (5 s on, 5 s off) for 5 min (total time). The lysate was subjected to centrifugation (5,000 × g at 4°C) for 15 min to remove cellular debris, and the cleared supernatants containing the hexa-His-tagged AAC(2')-Ia [His-AAC(2')-Ia] were mixed with Ni2+-NTA agarose and incubated with shaking for 1 h at 4°C. The slurry was then poured into a 10- by 1-cm disposable column and washed with 8 column volumes of the wash buffer (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole). The enzyme was eluted with the addition of 250 mM imidazole to the wash buffer.
For desalting and further purification, the isolated AAC(2')-Ia was applied to a Superdex 75 column that was equilibrated in 10 mM ammonium acetate at a flow rate of 1.0 ml/min. The eluted enzyme was determined to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and fractions were found to be stable at 4°C for approximately 2 weeks. This procedure typically yielded 31 mg of homogeneous enzyme per liter of cell culture.Enterokinase digestion. Samples (0.53 nmol) of purified His-AAC(2')-Ia in 100 µl of 10 mM ammonium acetate (pH 6.0) were digested with 1 µg (16.5 U) of enterokinase for 16 h at 20°C. The released hexa-His tag was removed by adsorption to 10 µl of Ni2+-NTA agarose at 4°C, and the digested enzyme was recovered from the supernatant after centrifugation at 13,000 × g.
Determination of enzyme activity. Routine assays for His-AAC(2')-Ia activity were conducted using the phosphocellulose binding assay (2) with 200 µM gentamicin and 160 µM [3H]acetyl-CoA (4 µCi/µmol) as substrates. A microtiter plate assay was used for the kinetic analysis of His-AAC(2')-Ia, monitoring in situ the time course of free CoA release from 80 µM acetyl-CoA by titration with 2 mM 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) (19) with 1 to 235 µM tobramycin as a substrate in 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 6.0) buffer containing 1 mM EDTA (200-µl final volume) at 25°C. Reaction mixtures without the addition of enzyme served as controls for the slow spontaneous hydrolysis of DTNB.
pH and temperature optima. Enzyme stability studies were performed by incubating the enzyme preparations [His-AAC(2')-Ia and AAC(2')-Ia following enterokinase digestion] at the desired temperature and pH for 90 min prior to determining residual activity against 64 µM tobramycin in 50 mM MES (pH 6.0) at 20°C using the microtiter plate assay. The buffers used for the pH studies were 50 mM sodium acetate, pH 3.7 to 5.7; 50 mM MES, pH 5.1 to 7.1; 50 mM MOPS (morpholinepropanesulfonic acid), pH 6.1 to 8.1; and 50 mM Tris-HCl, pH 7.1 to 9.1. The optimal temperature for activity was determined by preincubating the substrate mixture at the desired temperature for at least 10 min prior to addition of enzyme.
Enzyme kinetics.
The microtiter plate assay was used for the
kinetic analysis of His-AAC(2')-Ia. Various concentrations of kanamycin
sulfate, gentamicin sulfate, tobramycin, dibekacin, neomycin sulfate,
butirosin, and netilimicin (Fig. 1) were
added to the DTNB assay mixture as described above, and reactions were
initiated by the addition of 200 nM purified enzyme (final
concentration). The concentration of acetyl-CoA was held at 80 µM for
the analysis of the different aminoglycosides, while 64 µM tobramycin
was used as a cosubstrate for the determination of the kinetic
parameters for acetyl-CoA. DTNB was replaced by 4,4'-dithiodipyridine
and monitoring was at 324 nm (extinction coefficient, 19,800 M
1 cm1) for assays with
kanamycin due to the presence of interfering contaminants in the
antibiotic preparation. The Michaelis-Menten parameters were obtained
by graphing the initial velocity data using Enzfitter (Biosoft,
Cambridge, United Kingdom).
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Miscellaneous methods. MICs of the different aminoglycosides used in this study against P. stuartii PR50 were determined in LB broth containing twofold dilutions of the antibiotics between 0.1 and 128 µg/ml. SDS-PAGE was conducted using the method of Lamella (7), while protein concentrations were determined by the method of Smith (17) using the micro-bicinchoninic acid protein assay reagent (BCA; Pierce, Rockford, Ill.).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Subcloning and expression of aac(2')-Ia Originally the aac(2')-Ia gene was subcloned from pSCH4500 (15) into pBAD24 for its overexpression. However, expression trials of transformed E. coli BL21(DE3) giving E. coli AJC103 revealed that low and insufficient levels of enzyme were being produced for the purposes of this and future studies. Therefore, aac(2')-Ia was subcloned into pET30a(+), an overexpression vector under the control of the strong IPTG-inducible T7 promoter, to give pACKF2. This construct consisted of the open reading frame encoding AAC(2')-Ia with an N-terminal hexahistidine tag, which aided in its subsequent purification. Sequencing of pACKF2 confirmed the presence of the complete aac(2')-Ia gene in pET30a(+) (data not shown).
E. coli BL21(DE3) was transformed with pACKF2 to provide
strain AJC105. The MIC of gentamicin against AJC105 was determined to
confirm both the expression and activity of His-AAC(2')-Ia. Strain
AJC105 required an MIC of 16 µg of gentamicin per ml, which was 64 times higher than that for the parent E. coli strain
harboring only pET30a(+) (0.25 µg/ml) and four times higher than that
for the original E. coli transformant, strain
DH5/pSCH4500 (4 µg/ml) (15), from which we obtained
the aac(2')-Ia gene. The overexpression and activity of
His-AAC(2')-Ia were also confirmed using the phosphocellulose binding
assay (data not shown).
Production and isolation of AAC(2')-Ia.
After induction of
E. coli AJC105 with IPTG, 379 mg of total protein per liter
of cell culture supernatant was detected (Table 2). Initial purification using
Ni2+-NTA agarose to bind the His-tagged protein
resulted in the isolation of a total of 41 mg of protein. Gel exclusion
chromatography on Superdex was then used to further purify the enzyme
of contaminating proteins which nonspecifically adsorbed to the
affinity matrix and to remove the imidazole from the samples. This
final gel permeation step yielded 31 mg of protein judged to be
approximately 98% homogeneous by SDS-PAGE (Fig.
2). Based on the increase in specific
activity using the microtiter plate assay, a ninefold purification
was achieved, with a total recovery of greater than 70% of the
original activity.
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Temperature dependence of His-AAC(2')-Ia.
The purified enzyme
was assayed for activity against tobramycin in 25 mM ammonium acetate
buffer (pH 5.0) at different temperatures ranging from 5 to 65°C.
Surprisingly, the enzyme appeared to be relatively temperature
sensitive, as optimal activity was observed at 20°C (Fig.
3). The activity sharply declined on
either side of this temperature optimum, and less than 10% of maximal
activity was detected at temperatures greater than 40°C. In keeping
with these results, His-AAC(2')-Ia was found to also be thermolabile. After incubation of the enzyme at pH 5 for 1 h at different
temperatures and then assaying for residual activity at 20°C, maximal
activity was found to be maintained at 15°C, with only approximately
60% being retained at 30°C (Fig. 3). The activity of His-AAC(2')-Ia dramatically declined to less than 10% at higher temperatures. These
experiments were repeated with AAC(2')-Ia, prepared by enterokinase digestion of His-AAC(2')-Ia, and identical results were obtained (data
not shown).
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) and stability (
). For activity determinations, enzyme (200 nM
final concentration) was assayed against 64 µM tobramycin in 50 mM
MES (pH 6.0) at the indicated temperatures. For stability studies,
enzyme (200 nM) in the same buffer was incubated at the different
temperatures for 90 min prior to being assayed against 64 µM
tobramycin at 20°C.
-lactamase (see, e.g.,
reference 8). The presence of the N-terminal His tag was
not considered to be responsible for the destabilization because its
complete removal by enterokinase digestion did not alter the observed
temperature dependence.
pH dependence of His-AAC(2')-Ia.
Incubation of
His-AAC(2')-Ia at various pH values showed that the enzyme was
most stable at pH 8.1 (Fig. 4). The
enzyme retained at least 60% of its maximal activity between pH 6.1 and 8.6, but incubation beyond these pH values rendered the enzyme
virtually inactive.
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); 50 mM MOPS, pH 6.1 to 8.1 (
); and 50 mM Tris-HCl, pH 7.1 to 9.1 (
). (A) Enzyme (200 nM
final concentration) was assayed against 64 µM tobramycin at 20°C
at the pH values indicated using the microtiter plate assay. (B) Enzyme
(200 nM) was incubated in the different buffers at 20°C for 90 min
prior to being assayed against 64 µM tobramycin in 50 mM MES, pH
6.0.
Kinetic parameters of AAC(2')-Ia.
As previously documented
with crude preparations of P. stuartii AAC(2')-Ia
(15), His-AAC(2')-Ia demonstrated a broad
specificity toward substrates with an amino group at the 2' position of
aminoglycosides. Seven aminoglycosides were selected for kinetic
analysis using the microtiter plate assay, based on free CoA-SH
titration, of Williams and Northrop (19). The kinetic
parameters of His-AAC(2')-Ia for these different substrates
were determined assuming one active site per monomer and are presented
in Table 3. Using a fixed acetyl-CoA concentration of 80 µM, which was 10-fold higher than its
determined Km, gentamicin was found to be
the best aminoglycoside substrate with respect to catalytic efficiency
(kcat/Km),
whereas neomycin was the poorest. These differences appeared to
result from greater differences in turnover number
(kcat), which varied by almost 10-fold
compared to affinity, as reflected by the
Km values differing by less than 4-fold.
This was somewhat surprising and is very difficult to rationalize in
the absence of mechanistic details of the reaction pathway.
Nonetheless, these data indicated that His-AAC(2')-Ia was 1 and
2 orders of magnitude more efficient than the AAC(2') enzymes from
different Mycobacterium species (6) and
E. faecium AAC(6')-Ii, respectively. In general,
however, none of the aminoglycosides tested were considered to be good substrates for the enzyme, as the efficiency constants for each were
well below both the diffusion limit of 108 to
109
M1 s1.
|
Correlation between kinetic parameters and MIC.
The finding
that the aminoglycosides are relatively poor substrates for
His-AAC(2')-Ia is in keeping with our previous observations that AAC(2')-Ia has a housekeeping function in P. stuartii, namely, the O acetylation of peptidoglycan
(9-11), and that conferring resistance to aminoglycosides
is simply a fortuitous (to the bacterium) side reaction
(15). Likewise, it is suspected that AAC(6')-Ii may
play a physiological role in E. faecium, the nature of which is presently unknown (20). Both Piepersberg et al. (12)
and Uduo et al. (18) had questioned over 10 years ago
whether or not aminoglycoside acetyltransferase had a metabolic
function, while Ho et al. (6) more recently went as
far as to question whether AAC(2') enzymes in
Mycobacterium species play any significant role in
conferring resistance to aminoglycosides, even though they are
apparently universally present in the mycobacteria (1). This view is actually supported by an analysis of the correlation between MICs of the various aminoglycosides against P. stuartii and the kinetic parameters obtained with
His-AAC(2')-Ia. It has been argued that an efficient
detoxifying enzyme should be maximally effective at low antibiotic
concentrations (13), and such efficiency can be
experimentally demonstrated by a positive correlation between antibiotic resistance of a bacterial stain (as reflected by MICs) and
values of
kcat/Km
(the maximal rate at sub-Km substrate
concentrations) obtained with the isolated putative resistance factor.
Such was demonstrated with AAC(6')-IV from E. coli W677
(13), but as depicted in Fig.
5, the aminoglycosides tested against
P. stuartii PR50 and His-AAC(2')-Ia did not exhibit
this relationship. There appeared to be no correlation between the
kinetic parameters
kcat/Km and kcat and MICs of the
different aminoglycosides against P. stuartii PR50. A
similar situation was observed with ACC(6')-Ii and
aminoglycoside susceptibility with E. faecium
(20). With the latter enzyme, however, a positive
correlation was noted between MIC and
kcat, the catalytic rate at saturating
substrate concentrations, indicating that the cell would have to be
overrun with antibiotic before an effective resistance was mounted
by AAC(6')-Ii, which would obviously be too late.
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, dashed line) (r = 0.009773).
Concluding remarks. The O acetylation of peptidoglycan occurs as a maturation event, after the transglycosylation and transpeptidation of precursor subunits into the existing sacculus. This would suggest a periplasmic localization of AAC(2')-Ia by the cell and hence its indirect use of acetyl-CoA as a cosubstrate. We have proposed that an intrinsic membrane protein serves to translocate acetate from cytoplasmic pools of acetyl-CoA to the external surface of the cytoplasmic membrane and make it available to the peptidoglycan O-acetyltransferase(s) (5). The availability of highly purified AAC(2')-Ia will permit us to design experiments to test this hypothesis.
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ACKNOWLEDGMENTS |
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We thank P. Rather and L. Burrows for the kind gifts of bacterial strains and plasmids.
These studies were supported by an operating grant to A.J.C. from the Medical Research Council.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Phone: (519) 824-4120, ext. 3361. Fax: (519) 837-1802. E-mail: aclarke{at}micro.uoguelph.ca.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Antimicrobial Agents and Chemotherapy, August 2001, p. 2238-2244, Vol. 45, No. 8
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2238-2244.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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