Departamento de Química Biológica, Area Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK) Rosario, Argentina
Correspondence
Oscar A. Roveri
oroveri{at}fbioyf.unr.edu.ar
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ABSTRACT |
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INTRODUCTION |
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Azospirillum spp. are widely distributed soil nitrogen-fixing bacteria that live in close proximity to plant roots, and they play an important role in the promotion of plant growth (Steenhoudt & Vanderleyden, 2000). Wild-type strains of Azospirillum lipoferum and Azospirillum brasilense were found to be naturally resistant to penicillins, a resistance that was attributed to
-lactamase activity (Franche & Elmerich, 1981
). We have previously reported conclusive evidence for the presence of an inducible
-lactamase in A. lipoferum RG20 (Boggio et al., 1989
). In this study, we provide experimental evidence demonstrating that this enzyme is responsible for the natural resistance of A. lipoferum RG20 to
-lactam antibiotics. We also report the purification and catalytic properties (substrate, inhibitor and pH profiles) of the benzylpenicillin-induced A. lipoferum RG20
-lactamase.
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METHODS |
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Susceptibility tests.
MIC values were determined in test tubes filled with 3 ml of LuriaBertani (LB) medium (Ausubel et al., 1993), which were inoculated with 0·1 ml of an exponential-phase culture (5x107 cells ml-1). The antibiotics were added at various concentrations and the cells were incubated for 18 h at 37 °C.
Induction tests.
The -lactam compounds to be tested (100 µg ml-1) were added to a culture in exponential phase (OD540 value of between 0·8 and 0·9). Cells were harvested 1 h later by centrifugation at 4000 g for 10 min at 4 °C and washed with 100 mM potassium phosphate buffer (pH 7·0). The suspension of washed cells was immersed in an icewater slurry bath and sonicated (two 30 s pulses) with an MSE 150 Ultrasonic Desintegrator. The sonicate was centrifuged at 12 000 g for 15 min at 4 °C to remove cells debris.
-Lactamase activity, expressed as mU per 10-9 cells, was determined spectrophotometrically in the supernatant using nitrocefin (
482=15 900 M-1 cm-1) as substrate (O'Callaghan et al., 1972
). The activity determined in extracts from cells grown in the absence of antibiotic was 20 mU per 10-9 cells.
Enzyme assays.
-Lactamase activity was measured spectrophotometrically in 1 ml quartz cuvettes at 30 °C (Waley, 1974
). The differential absorption coefficients (unhydrolysed minus hydrolysed) for the different antibiotics used were: benzylpenicillin,
230=-1090 M-1 cm-1; ampicillin,
233=-780 M-1 cm-1; carbenicillin,
232=-700 M-1 cm-1; cephaloridine,
260=-9490 M-1 cm-1; cefotaxime,
262=-5990 M-1 cm-1; cephalothin,
263=-7380 M-1 cm-1; oxacillin,
263=290 M-1 cm-1. The reaction was started by the addition of the enzyme to a medium containing 100 mM sodium phosphate buffer (pH 7·0) and appropriate concentrations of the antibiotics. One enzyme unit (U) is defined as the amount of enzyme that hydrolysed 1 µmol of substrate in 1 min under assay conditions.
Purification of the enzyme.
A. lipoferum RG20 was grown in 6 l Erlenmeyer flasks containing 1·5 l of LB medium in a New Brunswick Giratory Water Bath Shaker (250r.p.m.) at 37 °C. Benzylpenicillin (100 µg ml-1) was added when the OD540 value reached between 0·8 and 0·9. One hour later the cells were harvested by centrifugation at 4000 g for 10 min at 4 °C and washed with 100 mM potassium phosphate buffer (pH 7·0). All further operations were carried out at 4 °C. Washed cells were resuspended in 20 % sucrose and 30 mM Tris/HCl (pH 7·0), treated with lysozyme as described (Lindström et al., 1970) and centrifuged at 16 000 g for 15 min. The pellet was resuspended in cold water and the suspension was centrifuged at 27 000 g for 30 min. The supernatant (crude extract) was precipitated with ammonium sulphate. Most of the activity was recovered in the 4085 % (NH4)2SO4 saturation fraction, which was resuspended in 10 mM potassium phosphate buffer (pH 7·0), dialysed against the same buffer containing 15 % (v/v) glycerol (buffer A), and applied to a CM-Sephadex column (1·8x16 cm) equilibrated with buffer A. The column was washed with buffer A plus 50 mM NaCl. A 50500 mM linear NaCl gradient was applied and
-lactamase activity was recovered in the fractions corresponding to about 200 mM NaCl. The more-active fractions were combined, dialysed against 20 mM Tris/HCl buffer (pH 7·0) and 15 % (v/v) glycerol (buffer B) and applied to a Cibacron Blue 3GA-Agarose column (1·5x10 cm) equilibrated with the same buffer. The column was washed with buffer B plus 100 mM NaCl and subsequently developed with a 100800 mM linear gradient of NaCl.
-Lactamase eluted at about 200 mM NaCl. The purified enzyme was concentrated using Centriprep 10 concentrators (Amicon) and stored at -20 °C in 20 mM Tris/HCl buffer (pH 7·0) containing 15 % (v/v) glycerol.
Enzyme activity was monitored during purification as indicated in the Enzyme assays' section of Methods using 1 mM benzylpenicillin as substrate.
Electrophoresis.
SDS-PAGE was performed in 12·5 % vertical slab mini-gels by the method of Laemmli (1970). Gels were stained with Coomasie brilliant blue R250.
Isoelectric focussing.
Analytical isoelectric focussing was performed in a 111 Mini IEF Cell apparatus (Bio-Rad) on polyacrylamide gels in the presence of ampholines in the pH range 810·5. Broad-range pI markers from Pharmacia were used. Gels were stained with Coomasie brilliant blue R250. The -lactamase band was identified by placing filter paper soaked in a 100 µM solution of nitrocefin on the gel (Matthew et al., 1975
).
Molecular mass determination.
The apparent molecular mass of the denatured purified -lactamase was determined from the mobility of the protein on SDS-PAGE. Molecular mass markers were BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20·1 kDa) and
-lactalbumin (14·4 kDa) (Pharmacia).
Estimation of kinetic parameters.
Initial velocities at different substrate concentrations were determined in duplicate as indicated in the Enzyme assays' section of Methods. Km and Vmax values were estimated by fitting the experimentally determined values to the MichaelisMenten equation using a non-linear regression procedure. An apparent molecular mass of 31 000 Da was used to calculate kcat values.
Inhibitor studies.
Initial velocities were determined at different fixed benzylpenicillin concentrations and variable inhibitor concentrations. The reaction was started by addition of the enzyme and was continuously monitored over 1 min as indicated in the Enzyme assays' section of Methods. v0/vi Values (where v0 is the initial velocity in the absence and vi is the initial velocity in the presence of inhibitor) were plotted against inhibitor concentration [I] and analysed as described by Roveri (1985). Linear inhibitors yield linear plots whose slopes depend on substrate concentration in a characteristic manner that makes possible the diagnosis of the inhibitor type and the estimation of Ki values.
General.
Protein determinations were carried out according to Lowry et al. (1951) using BSA as the standard, the concentration of which was determined spectrophotometrically (A279=6·67 cm-1 for a 1 % solution; Foster & Sterman, 1956
).
Concentration of cells was determined by measuring the optical density of a cell suspension at 540 nm (OD540=2 for 109 cells ml-1).
Chemicals.
Nitrocefin was a generous gift from Glaxo. Antibiotics and -lactamase inhibitors were obtained as follows: benzylpencillin, ampicillin, oxacillin, cephalothin, cloxacillin, cephalosporin C and cephaloridine, Sigma; carbenicillin and sulbactam, Pfizer; clavulanic acid and cephazolin, Smith Kline Beecham; cefotaxime, Aventis; aztreonam, Merck Sharp Dohme. 6-
-Bromopenicillanic acid was kindly synthesized, according to Pratt & Loosemore (1978)
, by Dr O. A. Mascaretti (IQUIOS, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina). Lysozyme and Cibacron Blue 3GA-Agarose were from Sigma. Other chemicals were of analytical grade.
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RESULTS AND DISCUSSION |
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Crude extracts obtained from benzylpenicillin-induced A. lipoferum cells were analysed by isoelectric focussing. A single intense band that focussed at pH 9·3 was detected when the gel was specifically stained with nitrocefin as indicated in Methods.
6--Bromopenicillanic acid, a known irreversible inhibitor of serine
-lactamases (Pratt & Loosemore, 1978
), abolished the resistance of A. lipoferum to
-lactam antibiotics. 6-
-Bromopenicillanic acid (0·5 µg ml-1) strongly decreased MIC values for benzylpenicillin (from >1000 to 0·8 µg ml-1), cephalosporin C (from >1000 to 0·4 µg ml-1) and oxacillin (from 200 to 0·4 µg ml-1). These results suggest that A. lipoferum resistance to
-lactam antibiotics is mainly due to its inducible
-lactamase.
Purification of -lactamase
-Lactamase from A. lipoferum cells induced by benzylpenicillin was purified as indicated in Methods. A final purification of 518-fold was achieved (Table 1
). The purified enzyme had a specific activity of 2155 U mg-1 and was homogeneous on SDS-PAGE and isoelectric focussing.
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The activity of the enzyme was affected neither by exhaustive dialysis against 100 mM potassium phosphate buffer (pH 7·0), 15 % (v/v) glycerol and 100 mM EDTA nor by the addition of 0·25 mM ZnSO4 to the reaction medium. However, the initial velocity of benzylpenicillin hydrolysis catalysed by the -lactamase of A. lipoferum was inhibited by clavulanic acid. The inhibition was competitive (Ki=3·1±0·2 µM) with respect to benzylpenicillin (Fig. 2
). Sulbactam also behaved as a linear competitive inhibitor with a Ki value of 46±9 µM (data not shown). Therefore, the
-lactamase of A. lipoferum does not belong to the group 3 metalloenzymes of the BushJacobyMedeiros scheme because it was not inhibited by EDTA and did not require Zn2+. More likely, it is an active-site serine enzyme that can be included in group 2 of the scheme since it was inhibited by clavulanic acid and sulbactam known active-site-directed
-lactamase inhibitors.
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kcat decreased with increasing pH values (Fig. 3a) according to Equation 1
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Equation 2 describes the kinetic behaviour of the system shown in Scheme II (Fig. 5). According to Scheme II, two ionic forms of the free enzyme (EHn+1 and EHn) could participate in productive binding of substrate, whereas a third one (EHn-1) would be unable to productively bind substrate. The estimated catalytic efficiency of EHn is two orders of magnitude higher than that of EHn+1 (Scheme II and Fig. 3b
). A similar pH-rate profile has been described for the RTEM-2
-lactamase (Knap & Pratt, 1991
). However,
-lactamase from A. lipoferum differs from the RTEM-2
-lactamase in the fact that pK1(E)>pK2(E), which indicates that proton binding is positively cooperative.
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In summary, the high natural resistance of A. lipoferum RG20 to -lactam antibiotics is due to the production of an inducible
-lactamase with an interesting pH-rate dependence and substrate and inhibitor profiles that agree well with those of group 2b
-lactamases of the BushJacobyMedeiros scheme.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 2 August 2002;
revised 5 November 2002;
accepted 7 November 2002.
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