| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-lactamase responsible for the resistance of Azospirillum lipoferum to -lactam antibiotics
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
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
-Bromopenicillanic acid, an irreversible inhibitor of serine--lactamases, completely abolished this resistance. A -lactamase was purified 518-fold from a cell-free extract of A. lipoferum RG20. A single band on SDS-PAGE (apparent molecular mass 31 000 Da) and on isoelectric focussing (pI9·35) was observed with the purified protein. The enzyme hydrolysed benzylpenicillin, ampicillin, cephalothin and cephaloridine with comparable kcat values and catalytic efficiencies. However, carbenicillin and cefotaxime were hydrolysed with significantly lower kinetic parameters and oxacillin was hydrolysed at a rate 100 times slower. The purified -lactamase was inhibited by clavulanic acid and sulbactam but not by EDTA or aztreonam. Its substrate and inhibitor profiles are consistent with those of the broad-spectrum -lactamases inhibited by clavulanic acid (group 2b of the Bush–Jacoby–Medeiros scheme). The effect of pH on kcat and Km values for benzylpenicillin hydrolysis was studied. The dependence of kcat on pH suggests that the enzyme–substrate (ES) complex must be in at least three protonation states: two with kcat values equal to 2800 and 1450 s-1 and a third inactive one [pK1(ES) 4·7 and pK2(ES) 7·9]. Similarly, the dependence of kcat/Km on pH can be explained by postulating that the enzyme free form can be at least in three different protonation states: two of them with kcat/Km values equal to 2·7x106 and 3·7x108 M-1 s-1 and a third one unable to productively bind substrate. Interestingly, the dependence of kcat/Km on pH is consistent with positive cooperativity for proton binding to the enzyme free form [pK1(E) 8·5 and pK2(E) 7·2].
Present address: Instituto de Biología Molecular y Celular de Rosario (CONICET-UNR), División Biología Molecular, Suipacha 531, (S2002LRK) Rosario, Argentina. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
-lactamases (EC 3.5.2.6) is the most prevalent mechanism of bacterial resistance to -lactam antibiotics (Philippon et al., 1998
). These enzymes were detected in bacteria before the widespread therapeutic use of penicillin (Abraham & Chain, 1940) and their presence in many pathogenic and non-pathogenic micro-organisms – including phototrophic bacteria – has been reported (Baumann et al., 1989; Bush et al., 1995). It has been proposed that -lactamases are evolutionarily related to cell-wall biosynthetic enzymes, viz. the penicillin-binding proteins. It is presumed that some micro-organisms evolved -lactamases in order to overcome the challenge of -lactam antibiotics, which were being exuded in the environment by their competitors (Massova & Mobashery, 1998).
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.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Susceptibility tests.
MIC values were determined in test tubes filled with 3 ml of Luria–Bertani (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 ice–water 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 40–85 % (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 50–500 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 100–800 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 8–10·5. 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 Michaelis–Menten 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.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
-lactam antibiotics
1000 µg ml-1), whereas it was more susceptible to oxacillin and cloxacillin (MIC=200 µg ml-1). The ability of these antibiotics to induce -lactamase activity was tested. The activity was increased 100- to 300-fold by the antibiotics with higher MIC values and 20-fold by the antibiotics with lower MIC values. 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.
|
). Since a value not significantly different (27·5 kDa) was obtained when the apparent molecular mass of the native protein was determined by Sephadex G-100 gel filtration it can be concluded that the -lactamase of A. lipoferum is a monomeric enzyme. A pI value of 9·35 was determined for the enzyme by isoelectric focussing.
|
-lactamases based on their substrate and inhibitor profiles (Bush, 1989; Bush et al., 1995). This scheme distinguishes four functional groups of -lactamases. In order to include the A. lipoferum -lactamase in one of these functional groups, we determined the Zn2+ requirement of the enzyme, the effect of inhibitors such as EDTA, clavulanic acid, sulbactam and aztreonam, and the kinetic parameters for several -lactam antibiotics.
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 Bush–Jacoby–Medeiros 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.
|
-lactamase with the Km and kcat values shown in Table 2. The enzyme behaved as a broad-spectrum -lactamase, since it hydrolysed benzylpenicillin, ampicillin, cephalothin and cephaloridine with comparable kcat values. Carbenicillin was hydrolysed with a kcat value 10 times smaller than that of the aforementioned antibiotics. Oxacillin was hydrolysed at a rate 100 times slower than benzylpenicillin. Extended-spectrum antibiotics were not good substrates or ligands for the enzyme: (i) cefotaxime was hydrolysed with catalytic efficiency <5 % that estimated for benzylpenicillin; (ii) aztreonam was not hydrolysed at an appreciable rate by the enzyme; and (iii) 1 mM aztreonam did not inhibit nitrocefin hydrolysis. On these grounds, the enzyme can be allocated to group 2b of the Bush–Jacoby–Medeiros scheme. Accordingly, its molecular mass (31 kDa) and isoelectric point (9·35) fall within the range of values (19–36 kDa and 4·9–9·5, respectively) reported for group 2b enzymes (Bush et al., 1995).
|
![{els]](/math/els.gif)
5 and (ii) a fast and irreversible inhibition was observed when the enzyme was pre-incubated with benzylpenicillin at pH 9.
kcat decreased with increasing pH values (Fig. 3a) according to Equation 1
 |
) and Fig. 3a]. Since no decay of activity was observed in the acid region, it can be presumed that the pK value for the general base that has been suggested to play a role in the reaction mechanisms of class A and C -lactamases (Lamotte-Brasseur et al., 1999; Page & Laws, 1998) is lower than 4·7. A similar behaviour has been reported for the kcat dependence on pH of the -lactamase of Streptomyces albus G (Brannigan et al., 1991).
|
|
) according to Equation 2:
 |
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.
|
-lactamase described here is near the diffusion control limit as it would be expected for a ‘perfect catalyst’. However, the catalytic efficiency for benzylpenicillin hydrolysis reached an experimental maximum (4x107 M-1 s-1) around pH 8·0, which is one order of magnitude lower than that estimated for EHn. Therefore, at the optimum pH only 10 % of the enzyme molecules would be in the ionic form with higher catalytic efficiency.
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 Bush–Jacoby–Medeiros scheme.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Ausubel, F. A., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1993). Current Protocols in Molecular Biology. New York: Greene Publishing & Wiley Interscience.
Baumann, M., Simon, H., Schneider, K.-H., Danneel, H.-J., Küster, U. & Giffhorn, F. (1989). Susceptibility of Rhodobacter sphaeroides to -lactam antibiotics: isolation and characterization of a periplasmic -lactamase (cephalosporinase). J Bacteriol 171, 308–313.
Boggio, S. B., Diaz Ricci, J. C., de Mendoza, D. & Roveri, O. A. (1989). Demonstration of the presence of an inducible -lactamase in Azospirillum lipoferum. Curr Microbiol 18, 33–35.[CrossRef]
Brannigan, J., Matagne, A., Jacob, F., Damblon, C., Joris, B., Klein, D., Spratt, B. G. & Frère, J.-M. (1991). The mutation Lys234His yields a class A -lactamase with a novel pH-dependence. Biochem J 278, 673–678.
Bush K. (1989). Characterization of -lactamases. Antimicrob Agents Chemother 33, 259–263.
Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for -lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 39, 1211–1233.[Medline]
Foster, J. F. & Sterman, M. D. (1956). Conformation changes in bovine serum albumin associated with hydrogen ion and urea binding. II. Hydrogen titration curves. J Am Chem Soc 78, 3656–3660.[CrossRef]
Franche, C. & Elmerich, C. (1981). Physiological properties and plasmid content of several strains of Azospirillum brasilense and A. lipoferum. Ann Microbiol (Paris) 132A, 3–18.
Knap, A. K. & Pratt, R. F. (1991). Inactivation of the RTEM-1 cysteine -lactamase by iodoacetate. The nature of the active-site functional groups and comparisons with the native enzyme. Biochem J 273, 85–91.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Lamotte-Brasseur, J., Lounnas, V., Raquet, X. & Wade, R. C. (1999). pKa calculations for class A -lactamases: influence of substrate binding. Protein Sci 8, 404–409.[Abstract]
Lindström, E. B., Boman, H. G. & Steele, B. (1970). Resistance of Escherichia coli to penicillins. VI. Purification and characterization of the chromosomally mediated penicillinase present in ampA-containing strains. J Bacteriol 101, 218–231.
Lowry, O. H., Rosebrough, A. L., Farr, L. H. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193, 3656–3660.
Massova, I. & Mobashery, S. (1998). Kinship and diversification of bacterial-penicillin binding proteins and -lactamases. Antimicrob Agents Chemother 42, 1–17.
Matthew, M., Harris, A. M., Marshall, M. J. & Ross, G. W. (1975). The use of analytical isoelectric focusing for detection and identification of -lactamases. J Gen Microbiol 88, 169–178.[Medline]
O'Callaghan, C., Morris, A., Kirby, S. M. & Shingler, A. H. (1972). Novel method for detection of -lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother 1, 282–288.
Page, M. I. & Laws, A. P. (1998). The mechanism of catalysis and the inhibition of -lactamases. Chem Commun 1998, 1609–1617.[CrossRef]
Philippon, A., Dussart, J., Joris, B. & Frère, J-M. (1998). The diversity, structure and regulation of -lactamases. Cell Mol Life Sci 54, 341–346.[CrossRef][Medline]
Pratt, R. F. & Loosemore, M. J. (1978). 6--Bromopenicillanic acid, a potent -lactamase inhibitor. Proc Natl Acad Sci U S A 75, 4145–4149.
Roveri, O. A. (1985). A new plot for the kinetic analysis of dead-end enzyme inhibitors. Biochem Int 11, 11–19.[Medline]
Steenhoudt, O. & Vanderleyden, J. (2000). Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol Rev 24, 487–506.[CrossRef][Medline]
Waley, S. G. (1974). A spectrophotometric assay of -lactamase action on penicillins. Biochem J 139, 789–790.[Medline]
Received 2 August 2002;
revised 5 November 2002;
accepted 7 November 2002.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
), 0·25 mM (
), 0·5 mM (
) and 1 mM (
). Slope values, estimated from the primary plots by linear regression, are plotted in the inset as a function of [Pen]. The line in the inset is the best fit obtained by non-linear regression analysis using the equation slope=KPen/{Ki(KPen+[Pen])}, which describes the behaviour of a linear competitive inhibitor (Ki=3·1±0·2 µM; KPen=0·50±0·07 mM).
; 
; pK1(ES)=4·7; pK2(ES)=7·9. (b) kcat/Km Values estimated at different pH values; the line is the theoretical behaviour described by Equation 2 with the following parameters: 
=3·7x108 M-1 s-1; 
=2·7x106 M-1 s-1; pK1(E)=8·5; pK2(E)=7·2.
