Proc. Nati. Acad. Sci. USA Vol. 73, No. 6, pp. 1816-1820, June 1976
Biochemistry
Altered penicillin-binding components in penicillin-resistant mutants of Bacillus subtilis (fluorography/affinity chromatography)
CHRISTINE E. BUCHANAN AND JACK L. STROMINGER The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138
Contributed by Jack L. Strominger, January 26, 1976
ABSTRACT Penicillin- (cloxacillin-) resistant mutants of Bacillus subtilis were isolated in a stepwise fashion and the five penicillin-binding components (PBCs) in each were examined to determine which of the proteins, if any, corresponds to the penicillin killing site. PBCs III and V were previously eliminated as the likely penicillin target. In the present work, PBC IV showed no change in sensitivity to cloxacillin in any of the resistant mutants isolated. PBC I did not change until the fifth-step mutant, in which it could not be detected by penicillin binding. Since PBC I did not bind penicillins that are lethal for this mutant, it also cannot be the lethal target. PBC II showed increased resistance to cloxaciffin in three discrete steps, i.e., in mutants 1, 4, and 5, accompanied by changes in its electrophoretic mobility. However, the sensitivity of PBC II to penicillin C changed very little. Correspondingly, the cloxacillinresistant mutants were unaltered in their sensitivity to penicillin G in vivo. Thus, of the five PBCs found in B. subtifis, PBC II is the most likely target for killing by penicillins.
killing site (9). Thus, the present study has concentrated on changes that might occur in PBCs I, II, and IV in penicillinresistant mutants of B. subtilis. MATERIALS AND METHODS Organism and Isolation of Mutants. B. subtilis strain Porton, resistant to 5 ,ug/ml of rifampicin and 200 /.g/ml of streptomycin, was used as the wild type. Penicillin sensitivity was determined by counting colonies on Antibiotic Medium 3 (Difco) agar plates containing various concentrations of the antibiotic at 30. To isolate a resistant mutant, a colony growing on a plate containing a high concentration of cloxacillin (E. R. Squibb & Sons, Inc.) was picked and purified by two rounds of single colony isolation. No mutagens were used. Cell-bound penicillinase activity was assayed as described (12). Preparation of Membranes and Isolation of PBCs. Membranes were prepared by grinding cells with glass beads as described previously (9). The penicillin-binding components were isolated by affinity chromatography as described (10) except the Sepharose 4B200 (Sigma) was coupled with 6-aminohexanoic acid (Aldrich) prior to 6-aminopenicillanic acid (6-APA) (Sigma) substitution. Penicillin Binding Assays. [14C]Penicillin G (AmershamSearle, 53 mCi/mmol) was bound to B. subtilis membranes as described (9). Sensitivity of the purified PBCs to a variety of penicillins was measured by first prebinding with an unlabeled penicillin at different concentrations and then adding a saturating concentration of [14C]penicillin G. The protocol was similar to that described elsewhere (8). The binding reaction was stopped by adding a 250-fold excess of unlabeled penicillin G, followed by precipitation of the protein with 80% acetone. The protein precipitate was dried with a stream of air, boiled in sample buffer (see below), and applied to a gel. Sodium Dodecyl Sulfate (NaDodSO4) Polyacrylamide Gel Electrophoresis. Tube gels were run as described by Weber and Osborn (13). The gels were 8 cm long and subjected to electrophoresis for 6 hr at 8 mA per gel. Discontinuous NaDodSO4 polyacrylamide slab gels (2 mm) were run as described (14, 15). The upper stacking gel was 3% acrylamide and the running gel was 7.5% acrylamide. The sample buffer contained 10% (vol/vol) glycerol, 5% mercaptoethanol, 3% NaDodSO4, 0.0625 M Tris-HCl, pH 6.8, and 0.002% bromphenol blue. Samples, suspended in 30 ;J of sample buffer and boiled for 5 min, were subjected to electrophoresis at 110 V for 3.5 hr. The gel was stained with Coomassie brilliant blue (16) for 2 hr at 370 followed by overnight destaining with 5% methanol and 7.5% acetic acid. "C-Labeled proteins in the dried slab gel were detected by fluorography (17). To quantitate the results, the x-ray film (after 1-4 days' exposure) was scanned with a double-beam recording microdensitometer manufactured by Joyce, Loebl and Co. Ltd.
Penicillin kills bacteria by inhibiting the crosslinking of the bacterial cell wall catalyzed by a transpeptidase (1, 2). The antibiotic is believed to be a substrate analogue of the enzyme and to inactivate the transpeptidase by forming a stable penicilloyl-enzyme intermediate (1). In fact, a similarity has been found in the concentration of penicillin required to kill bacteria and to saturate their penicillin killing sites (3-5). The interpretation has recently been complicated, however, by the demonstration of multiple penicillin-binding proteins in such organisms as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Bacillus stearothermophilus, and Bacillus cereus (6, 7). Not all of these proteins that specifically bind penicillin correspond to the lethal target of penicillin, nor does any one protein necessarily have the same sensitivity to each of a variety of penicillins and cephalosporins. The question remains, then, which (if any) of the different penicillin-binding components (PBCs) corresponds to the lethal target of penicillin? One approach to this problem is to determine which components become more resistant to penicillin upon mutation of the organism to greater penicillin resistance. Bacillus subtilis has five penicillin-binding components which can be easily isolated from solubilized membranes by affinity chromatography (8-10). Of the five, only PBC V has so far been found to possess enzymatic activity. It is the D-alanine carboxypeptidase and represents at least 80% of the penicillin-binding protein both in the membranes and in the purified PBCs (8, 11). Since this enzyme is not essential to normal growth in B. subtilis it is not a penicillin killing site (11). In addition, PBC III is not a killing site because it binds penicillin only at very high concentrations (9). On the other hand, PBCs I, II, and IV are all candidates for the penicillin killing site, since their antibiotic sensitivity profiles closely resemble that of the Abbreviations: PBC, penicillin-binding component; 6-APA, 6-ami-
nopenicillanic acid; NaDodSO4, sodium dodecyl sulfate. 1816
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Buchanan and Strominger
1817
Table 1. Sensitivity of wild type and mutant 5 to various penicillins
LD50 (gg/ml, 30°) NH,,
CH3
S
11.0
7.0
CjC H3
6-Aminopenicillanic acid
Mutant 5
WT
Structure
Penicillin
COOH O H
Benzylpenicillin (penicillin G)
CH NcKi OH3
0.012
CH,3005 COOH S
N
Cloxacillin
0005
OH3
S JCHC-N OHN N
CH3
18.0
0.1
OH3 CQOH O ON> OxacC N
OHI
C
Dicloxacillin c
NI
t..
S
-N cH O
S
C-N
Oxacillin
N
RESULTS Isolation of a Highly Penicillin Resistant Mutant. Highlevel penicillin resistance in bacteria develops in a stepwise fashion in the absence of penicillinase production (18). A single-step penicillin-resistant mutant shows only a small increase in penicillin resistance. Since small changes in the binding of penicillin to the individual PBCs are difficult to detect in vitro, and because sequential changes in PBCs might be of interest, a highly penicillin resistant mutant of B. subtilis was isolated by a series of stepwise increases in resistance. Cloxacillin was chosen as the selective penicillin because it is relatively resistant
OCH.3
N O
OH3
00
OH3 POOH
N
OH3 H3 O~~OOH
.
00
.
0.09
3.5
to the fl-lactamase of Bacillus species (19). The survival curves of the wild-type B. subtilis and five cloxacillin-resistant mutants (Fig. 1) illustrate the relatively small increase in cloxacillin resistance occurring in each step of the selection. Sensitivity of Mutant 5 to Other Penicillins. The mutant showed greatly increased resistance to oxacillin (Squibb) and dicloxacillin (Bristol Laboratories) as well as to cloxacillin (Table 1). Only a slight change in sensitivity to 6-APA or penicillin G was found. Mutant No. Wild 4 2 3 Type 1 , I I
5
10l4r
*
10-6 1.0 5.0 l0 40 Cloxacillin ,ug/ml FIG. 1. Survival curves of B. subtilis and the five mutants. The curves (from left to right) correspond to survival of the wild type, mutants 1, 2, 3, 4, and 5. The arrows indicate where a survivor was picked to be used for the isolation of the next step mutant. The plates were incubated at 30° to avoid selecting against any temperaturesensitive mutants that might occur. The LDWos (jg/ml), defined as the concentrations of cloxacillin required to reduce the number of colony-forming units by 50%, are as follows: wild type, 0.1; mutant 1, 0.38; mutant 2, 0.88; mutant 3, 2.7; mutant 4, 7.4; mutant 5, 18.0.
.05 .1
.5
FIG. 2 NaDodSO4 polyacrylamide gel of PBCs isolated by affinity chromatography from B. subtilis and five cloxacillin-resistant mutants. The protein above PBC Tin each sample is apparently unreduced penicillin-binding protein. In the absence of mercaptoethanol there was even a greater amount of protein in this area of the gel. Visible bands, other than the five PBCs, were not reproducible.
1818
Biochemistry: Buchanan and Strominger A
B
Proc. Natl. Acad. Sci. USA 73 (1976)
C
-I
-
11
III
-IV
WOW.
-v
FIG. 3. Isolated PBCs from B. subtilis wild type and mutant 5. (A) Mutant 5 PBCs. (B) Wild-type PBCs. (C) Mixture of the two PBC preparations. The altered mobility of PBC II from mutant 5 was evident in at least six different preparations.
Isolation of PBCs by Affinity Chromatography. Two interesting alterations were evident in stained NaDodSO4 gels of the PBCs purified by affinity chromatography (Fig. 2). In mutant 5, PBC I was not present. Either it did not bind to the 6-APA of the affinity column, e.g., because it was insensitive to penicillin, or the protein was absent in this mutant. The latter interpretation is supported by the observation that when [14C]penicillin G at high concentration was bound to membranes of mutant 5, no radioactive PBC I could be found, even in trace amount. A more subtle difference was found in the protein pattern of PBC II. PBC II appeared to have a slightly slower mobility in some of the mutants than in the wild type. An examination of PBC II in the series of mutants (Fig. 2) suggests there were several discrete changes in the mobility of PBC II, one evident 0
1
I
I
Wild Type 5 50
I
in mutants 4 and 5 as compared to mutant 3. In order to establish this change more firmly, the mutant 5 and wild-type samples were run separately in adjacent wells and together in a mixture on a slab gel (Fig. 3). The pattern clearly indicates that PBC II of mutant 5 is not identical to that of the wild type. Another slight alteration in PBC II may have occurred in mutant 1. Such small changes in electrophoretic mobility could be the result of a single amino acid change affecting the charge on the protein (20, 21). Examination of PBC II is further complicated by the fact that in some gels it appears to be two proteins. Further studies using higher resolution gel systems are necessary. PBCs III, IV, and V were present in all the organisms and no gross differences were seen among the preparations. PBC III consistently appeared as a doublet on slab gels, which was not observed previously or in the present work when the PBCs were run on tube gels. The lower half of the doublet was very resistant to penicillin, as was previously described (9). The higher molecular weight half of the doublet, on the other hand, was extremely sensitive to all penicillins tested in all of the mutants and the wild type. Kinetics of Cloxacillin Binding to PBCs I, II, and IV. Since radioactive cloxacillin was not available, cloxacillin binding was measured indirectly by competition for subsequent binding of labeled penicillin G (Fig. 4). The concentrations of cloxacillin (,gg/ml) used multiplied times the time of exposure to the antibiotic in minutes range from 0 to 500 in the experiment illustrated. PBC I, totally absent in mutant 5, was no more resistant to cloxacillin in mutant 4 than in the wild type. PBC IV was unaltered in its cloxacillin sensitivity in all of the mutants. PBC II was markedly increased in cloxacillin resistance in both mutants 4 and 5. Quantitative results from a number of such experiments are found in Fig. 5. The concentrations of cloxacillin (,ug/ml X min) required to reduce ['4C]penicillin G binding to each of the PBCs by 50% were estimated from these curves (Table 2). PBC I was unaltered in cloxacillin-resistant mutants 1-4. Its loss or its inability to bind penicillins, therefore, occurred in a single step. PBC II, on the other hand, appeared to undergo three changes in its resistance to cloxacillin, i.e., at mutants 1, 4, and 5. These changes were roughly similar to the changes in the resistance
Mutant 4 5 50 500
500
I
0
1
Mutant 5 5 50 500
Clox.
!
I_-
I
.
II III
IV -
Tq;
FIG. 4. Fluorograms of PBCs prebound with cloxacillin and labeled with [14C]penicillin G. Concentrations of cloxacillin are expressed as wg/ml [14C]penicillin G, except a slight amount by PBC V.
X min. Boiled controls bound no detectable
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Buchanan and Strominger
1819
14 XID
PBC31PBCIZ
PBC I
A .8
AT\ Uv.5 ~~~~~0o '0
~" 3 4
.6
,wr
A
~~~~
2
0
0 80~~~~~~~.
A
500 0 505 10 .5 1 2 -~~~~~~~nwr
0
5
WI
0
c~.2
0
3
A
X..
p~nu 0 .3I1z
-
uSUiV
I1
.i;,
5
5 I 1*v %0
2r-
10-
pAg/ml X min Cloxacillin FIG. 5. Kinetics of cloxacillin binding to the isolated PBCs of B. subtilis wild type and five cloxacillin-resistant mutants. Counts per minute ranged from 300 to 500 for each PBC when no cloxacillin was prebound. The points in the figure are the average of duplicate samples done on the same PBC preparation. In addition, each binding assay was performed on at least two different preparations of PBCs for each organism. These results were not averaged, but were plotted as separate points.
levels of the organisms (Fig. 1). PBC IV retained the same sensitivity to cloxacillin as the wild type in all five mutants. Preliminary measurements with oxacillin and dicloxacillin suggested a similar pattern of changes (data not shown). The affinity of the wild-type and mutant 5 PBCs for penicillin G was also measured. There was no significant change in the sensitivity of either PBC II or PBC IV from the mutant (Fig. 6). DISCUSSION It is clear from these studies that the selection of penicillinresistant mutants of Bacillus subtilis is accompanied by alterations in the penicillin-binding components of this organism. The change in sensitivity of PBC II to cloxacillin appeared to occur in three discrete steps, i.e., in mutants 1, 4, and 5. In mutant 4, and possibly in mutant 1, the change was accompanied by a change in electrophoretic mobility, while in mutant 5 the altered resistance was manifested only by an altered sensitivity to cloxacillin. Earlier a single-step mutant of B. subtilis showing a small change in resistance to 6-APA was isolated; in that mutant also, only PBC II exhibited a significant, reproducible decrease in its affinity for 6-APA (22). Direct proof of the relationship between the altered penicillin-binding component and altered penicillin sensitivity could be obtained by isolating a revertant in which both properties were reverted;
however, it is not possible to select for such a revertant. Alternatively, this problem might be resolved by genetic experiments involving transformation of B. subtilis. There was no apparent change in PBC II or any other PBC in mutants 2 and 3. Some other change must have occurred in these mutants, possibly a membrane change reflected in altered permeability rather than in an alteration in one of the PBCs. Increased production of a cell-bound penicillinase in any of the mutants was ruled out (data not shown). A remarkable fact is that the alteration in sensitivity of mutant 5 to cloxacillin was accompanied by little or no change in the organism's sensitivity to penicillin G. Correspondingly, there was little or no change in the sensitivity of PBC II to penicillin G despite the obvious alteration of the protein's electrophoretic mobility. This indicates that the active sites of penicillin-sensitive proteins may be modified in their sensitivity to one 0lactam antibiotic without any alteration in sensitivity to others. This fact may have implications for therapy with fl-lactam antibiotics and it is paralleled by wide differences in the sensitivity of PBCs in wild-type organisms to different f3-lactam antibiotics. A striking example of this is the extreme sensitivity of PBC II in E. coli to formamidino penicillin as compared to all other penicillins that have been examined (7). The loss of the penicillin-binding activity of PBC I that occurred during isolation of mutant 5 is an interesting finding.
PBC IV
PBC II 1.0
X 1.04 0 m .9
.96
CD .8 c = .7 O
.5
1
.4
Co
.7
.6 6
o-
.6 -
.3 '
Mutant 5
o .2
LL. I
Mutant 5
.5-
.4
U.3 \~~~~~~~~.
.
0 L.
.
-
Wild Type
'lI
0\
M\
.1
)ug/ml
x
.5
i
2
5
min penicillin G
FIG. 6. Kinetics of penicillin G binding to PBCs II and IV of the wild type and mutant 5.
1820
Biochemistry: Buchanan and Strominger
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 2. Cloxacillin resistance of PBCs Mutant
PBC I PBC II PBC IV
Wild type
1
2
3
4
5
0.54 1.75 0.8
1.08 3.8 0.92
1.55 2.65 1.62
1.04 2.18 1.18
0.66 11.4 0.76
72. 1.43
Resistance is defined by the amount of cloxacillin (Atg/ml x min) required to inhibit [14C]penicillin G binding by 50%. These values are read directly from the curves in Fig. 5.
This PBC could not be detected at all by the two techniques that are presently available (isolation of PBCs by affinity chromatography, or their labeling within the membranes with ['4C]penicillin G). However, it is conceivable that PBC I was present'in this mutant, but sufficiently altered in its resistance to penicillins that it escaped detection. When wild-type PBC I is purified, antibody to this protein might be prepared and crossreacting antigenic material sought. Remarkably, mutant 5 grows normally and, even under conditions of stress (growth in minimal media and growth at 470), no abnormality could be detected. The organism also sporulates and germinates normally. If this protein is actually absent in the mutant, then it appears to be unessential to its growth. Previous studies have indicated that PBC V is not essential for growth of B. subtilis (11). Conceivably the fine structure of the cell wall of mutant 5 might be altered by the absence of PBC I, but, in the case of elimination of PBC V from the wild type (by treatment with 6-APA), no alteration in cell wall structure could be found (23). One other possibility is that PBC I, which has the highest molecular weight of the five PBCs of B. subtilis, might be a precursor of one of the other PBCs, and that its absence in mutant 5 might simply be an acceleration of the rate of conversion; however, no evidence for this possibility has been obtained. A relationship between PBC I and PBC II might be suggested by the simultaneous occurrence in mutant 5 of an alteration in the resistance of PBC II and the disappearance of PBC I, since the isolation of a double mutant in a single step is improbable. In any case, PBC I is not likely to be a penicillin killing site, since mutant 5 is still quite sensitive to 6-APA and penicillin G, which have been shown not to bind to PBC I. In addition, PBC IV cannot be the lethal target of cloxacillin, at least, since it did not change in its affinity for cloxacillin in any of the five cloxacillin-resistant mutants. As discussed above, PBC I and PBC V are also eliminated. One of the proteins of the PBC III doublet is too insensitive to be the killing site, and the other is far too sensitive. Thus, all the evidence points to PBC
II as the likely target for killing of B. subtilis by f3-lactam antibiotics. The function of the various penicillin-binding components of B. subtilis remains an important unsolved problem. It may be imagined that these proteins are all enzymes that may participate in different aspects of cell wall synthesis, such as elongation, septation, and formation of "corners" of rods. Further study of mutants in which these PBCs are altered may shed light on these functions, and, certainly, isolation of the individual PBCs in a pure form is an important objective. The work was supported by research grants from the National Institutes of Health (AI-09152) and National Science Foundation (BMS71-01120). C.E.B. was a postdoctoral fellow of the American Cancer Society. 1. Tipper, D. J. & Strominger, J. L. (1965) Proc. Natl. Acad. Sci. USA 54, 1133-1141. 2. Wise, E. M. & Park. J. J. (1965) Proc. Nat!. Acad.- Sci. USA 54, 75-81. 3. Rowley, D., Cooper, P. D., Roberts, P. W. & Smith, E. L. (1950) Biochem. J. 46, 157-161. 4. Eagle, H. (1954) J. Exp. Med.- 99,207-226. 5. Edwards, J. R. & Park, J. T. (1969) J. Bacteriol. 99, 459-462. 6. Suginaka, H., Blumberg, P. M. & Strominger, J. L. (1972) J. Biol.
Chem. 247,5279-5288. 7. Spratt, B. G. & Pardee, A. B. (1975) Nature 254,516-517. 8. Blumberg, P. M. & Strominger, J. L. (1972) Proc. Natl. Acad. Sci. USA 69,3751-3755. 9. Blumberg, P. M. & Strominger, J. L. (1972) J. Brio. Chem. 247, 8107-8113. 10. Blumberg, P. M. & Strominger, J. L. (1974) in Methods in Enzymology, eds. Jakoby,W. B. & Wilchek, M. (Academic Press, New York), Vol. 34, pp. 401-405. 11. Blumberg, P. M. & Strominger, J. L. (1971) Proc. Natl. Acad. Sd. USA 68,2814-2817. 12. Hamilton, T. E. & Lawrence, P. J. (1975) J. Biol. Chem. 250, 6578-6585. 13. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244,4406-4412. 14. Laemmli, U. K. (1970) Nature 227,680-685. 15. Studier, F. W. (1973) J. Mol. Biol. 79,237-248. 16. Vesterberg, O. (1971) Biochim. Biophys. Acta 243,345-348. 17. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46,83-88. 18. Demerec, M. (1948) J. Bacteriol. 56,63-74. 19. O'Callaghan, C. H. & Muggleton, P. W. (1972) Cephalosporins and Penicillins. Chemistry and Biology, ed. Flynn, E. H. (Academic Press, New York), pp. 438-495. 20. Linn, T., Losick, R. & Sonenshein, A. L. (1975) J. Bacteriol. 122, 1387-1390. 21. Swaney, J. B., Vande Woulde, G. F. & Bachrach, H. L. (1974)
Anal. Biochem. 58,337-346.
22. Blumberg, P. M. (1973) Ph.D. Dissertation, Harvard University. 23. Sharpe, A., Blumberg, P. M. & Strominger, J. L. (1974) J. Bac-
teriol. 117,926-927.
Biochemistry
Altered penicillin-binding components in penicillin-resistant mutants of Bacillus subtilis (fluorography/affinity chromatography)
CHRISTINE E. BUCHANAN AND JACK L. STROMINGER The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138
Contributed by Jack L. Strominger, January 26, 1976
ABSTRACT Penicillin- (cloxacillin-) resistant mutants of Bacillus subtilis were isolated in a stepwise fashion and the five penicillin-binding components (PBCs) in each were examined to determine which of the proteins, if any, corresponds to the penicillin killing site. PBCs III and V were previously eliminated as the likely penicillin target. In the present work, PBC IV showed no change in sensitivity to cloxacillin in any of the resistant mutants isolated. PBC I did not change until the fifth-step mutant, in which it could not be detected by penicillin binding. Since PBC I did not bind penicillins that are lethal for this mutant, it also cannot be the lethal target. PBC II showed increased resistance to cloxaciffin in three discrete steps, i.e., in mutants 1, 4, and 5, accompanied by changes in its electrophoretic mobility. However, the sensitivity of PBC II to penicillin C changed very little. Correspondingly, the cloxacillinresistant mutants were unaltered in their sensitivity to penicillin G in vivo. Thus, of the five PBCs found in B. subtifis, PBC II is the most likely target for killing by penicillins.
killing site (9). Thus, the present study has concentrated on changes that might occur in PBCs I, II, and IV in penicillinresistant mutants of B. subtilis. MATERIALS AND METHODS Organism and Isolation of Mutants. B. subtilis strain Porton, resistant to 5 ,ug/ml of rifampicin and 200 /.g/ml of streptomycin, was used as the wild type. Penicillin sensitivity was determined by counting colonies on Antibiotic Medium 3 (Difco) agar plates containing various concentrations of the antibiotic at 30. To isolate a resistant mutant, a colony growing on a plate containing a high concentration of cloxacillin (E. R. Squibb & Sons, Inc.) was picked and purified by two rounds of single colony isolation. No mutagens were used. Cell-bound penicillinase activity was assayed as described (12). Preparation of Membranes and Isolation of PBCs. Membranes were prepared by grinding cells with glass beads as described previously (9). The penicillin-binding components were isolated by affinity chromatography as described (10) except the Sepharose 4B200 (Sigma) was coupled with 6-aminohexanoic acid (Aldrich) prior to 6-aminopenicillanic acid (6-APA) (Sigma) substitution. Penicillin Binding Assays. [14C]Penicillin G (AmershamSearle, 53 mCi/mmol) was bound to B. subtilis membranes as described (9). Sensitivity of the purified PBCs to a variety of penicillins was measured by first prebinding with an unlabeled penicillin at different concentrations and then adding a saturating concentration of [14C]penicillin G. The protocol was similar to that described elsewhere (8). The binding reaction was stopped by adding a 250-fold excess of unlabeled penicillin G, followed by precipitation of the protein with 80% acetone. The protein precipitate was dried with a stream of air, boiled in sample buffer (see below), and applied to a gel. Sodium Dodecyl Sulfate (NaDodSO4) Polyacrylamide Gel Electrophoresis. Tube gels were run as described by Weber and Osborn (13). The gels were 8 cm long and subjected to electrophoresis for 6 hr at 8 mA per gel. Discontinuous NaDodSO4 polyacrylamide slab gels (2 mm) were run as described (14, 15). The upper stacking gel was 3% acrylamide and the running gel was 7.5% acrylamide. The sample buffer contained 10% (vol/vol) glycerol, 5% mercaptoethanol, 3% NaDodSO4, 0.0625 M Tris-HCl, pH 6.8, and 0.002% bromphenol blue. Samples, suspended in 30 ;J of sample buffer and boiled for 5 min, were subjected to electrophoresis at 110 V for 3.5 hr. The gel was stained with Coomassie brilliant blue (16) for 2 hr at 370 followed by overnight destaining with 5% methanol and 7.5% acetic acid. "C-Labeled proteins in the dried slab gel were detected by fluorography (17). To quantitate the results, the x-ray film (after 1-4 days' exposure) was scanned with a double-beam recording microdensitometer manufactured by Joyce, Loebl and Co. Ltd.
Penicillin kills bacteria by inhibiting the crosslinking of the bacterial cell wall catalyzed by a transpeptidase (1, 2). The antibiotic is believed to be a substrate analogue of the enzyme and to inactivate the transpeptidase by forming a stable penicilloyl-enzyme intermediate (1). In fact, a similarity has been found in the concentration of penicillin required to kill bacteria and to saturate their penicillin killing sites (3-5). The interpretation has recently been complicated, however, by the demonstration of multiple penicillin-binding proteins in such organisms as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Bacillus stearothermophilus, and Bacillus cereus (6, 7). Not all of these proteins that specifically bind penicillin correspond to the lethal target of penicillin, nor does any one protein necessarily have the same sensitivity to each of a variety of penicillins and cephalosporins. The question remains, then, which (if any) of the different penicillin-binding components (PBCs) corresponds to the lethal target of penicillin? One approach to this problem is to determine which components become more resistant to penicillin upon mutation of the organism to greater penicillin resistance. Bacillus subtilis has five penicillin-binding components which can be easily isolated from solubilized membranes by affinity chromatography (8-10). Of the five, only PBC V has so far been found to possess enzymatic activity. It is the D-alanine carboxypeptidase and represents at least 80% of the penicillin-binding protein both in the membranes and in the purified PBCs (8, 11). Since this enzyme is not essential to normal growth in B. subtilis it is not a penicillin killing site (11). In addition, PBC III is not a killing site because it binds penicillin only at very high concentrations (9). On the other hand, PBCs I, II, and IV are all candidates for the penicillin killing site, since their antibiotic sensitivity profiles closely resemble that of the Abbreviations: PBC, penicillin-binding component; 6-APA, 6-ami-
nopenicillanic acid; NaDodSO4, sodium dodecyl sulfate. 1816
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Buchanan and Strominger
1817
Table 1. Sensitivity of wild type and mutant 5 to various penicillins
LD50 (gg/ml, 30°) NH,,
CH3
S
11.0
7.0
CjC H3
6-Aminopenicillanic acid
Mutant 5
WT
Structure
Penicillin
COOH O H
Benzylpenicillin (penicillin G)
CH NcKi OH3
0.012
CH,3005 COOH S
N
Cloxacillin
0005
OH3
S JCHC-N OHN N
CH3
18.0
0.1
OH3 CQOH O ON> OxacC N
OHI
C
Dicloxacillin c
NI
t..
S
-N cH O
S
C-N
Oxacillin
N
RESULTS Isolation of a Highly Penicillin Resistant Mutant. Highlevel penicillin resistance in bacteria develops in a stepwise fashion in the absence of penicillinase production (18). A single-step penicillin-resistant mutant shows only a small increase in penicillin resistance. Since small changes in the binding of penicillin to the individual PBCs are difficult to detect in vitro, and because sequential changes in PBCs might be of interest, a highly penicillin resistant mutant of B. subtilis was isolated by a series of stepwise increases in resistance. Cloxacillin was chosen as the selective penicillin because it is relatively resistant
OCH.3
N O
OH3
00
OH3 POOH
N
OH3 H3 O~~OOH
.
00
.
0.09
3.5
to the fl-lactamase of Bacillus species (19). The survival curves of the wild-type B. subtilis and five cloxacillin-resistant mutants (Fig. 1) illustrate the relatively small increase in cloxacillin resistance occurring in each step of the selection. Sensitivity of Mutant 5 to Other Penicillins. The mutant showed greatly increased resistance to oxacillin (Squibb) and dicloxacillin (Bristol Laboratories) as well as to cloxacillin (Table 1). Only a slight change in sensitivity to 6-APA or penicillin G was found. Mutant No. Wild 4 2 3 Type 1 , I I
5
10l4r
*
10-6 1.0 5.0 l0 40 Cloxacillin ,ug/ml FIG. 1. Survival curves of B. subtilis and the five mutants. The curves (from left to right) correspond to survival of the wild type, mutants 1, 2, 3, 4, and 5. The arrows indicate where a survivor was picked to be used for the isolation of the next step mutant. The plates were incubated at 30° to avoid selecting against any temperaturesensitive mutants that might occur. The LDWos (jg/ml), defined as the concentrations of cloxacillin required to reduce the number of colony-forming units by 50%, are as follows: wild type, 0.1; mutant 1, 0.38; mutant 2, 0.88; mutant 3, 2.7; mutant 4, 7.4; mutant 5, 18.0.
.05 .1
.5
FIG. 2 NaDodSO4 polyacrylamide gel of PBCs isolated by affinity chromatography from B. subtilis and five cloxacillin-resistant mutants. The protein above PBC Tin each sample is apparently unreduced penicillin-binding protein. In the absence of mercaptoethanol there was even a greater amount of protein in this area of the gel. Visible bands, other than the five PBCs, were not reproducible.
1818
Biochemistry: Buchanan and Strominger A
B
Proc. Natl. Acad. Sci. USA 73 (1976)
C
-I
-
11
III
-IV
WOW.
-v
FIG. 3. Isolated PBCs from B. subtilis wild type and mutant 5. (A) Mutant 5 PBCs. (B) Wild-type PBCs. (C) Mixture of the two PBC preparations. The altered mobility of PBC II from mutant 5 was evident in at least six different preparations.
Isolation of PBCs by Affinity Chromatography. Two interesting alterations were evident in stained NaDodSO4 gels of the PBCs purified by affinity chromatography (Fig. 2). In mutant 5, PBC I was not present. Either it did not bind to the 6-APA of the affinity column, e.g., because it was insensitive to penicillin, or the protein was absent in this mutant. The latter interpretation is supported by the observation that when [14C]penicillin G at high concentration was bound to membranes of mutant 5, no radioactive PBC I could be found, even in trace amount. A more subtle difference was found in the protein pattern of PBC II. PBC II appeared to have a slightly slower mobility in some of the mutants than in the wild type. An examination of PBC II in the series of mutants (Fig. 2) suggests there were several discrete changes in the mobility of PBC II, one evident 0
1
I
I
Wild Type 5 50
I
in mutants 4 and 5 as compared to mutant 3. In order to establish this change more firmly, the mutant 5 and wild-type samples were run separately in adjacent wells and together in a mixture on a slab gel (Fig. 3). The pattern clearly indicates that PBC II of mutant 5 is not identical to that of the wild type. Another slight alteration in PBC II may have occurred in mutant 1. Such small changes in electrophoretic mobility could be the result of a single amino acid change affecting the charge on the protein (20, 21). Examination of PBC II is further complicated by the fact that in some gels it appears to be two proteins. Further studies using higher resolution gel systems are necessary. PBCs III, IV, and V were present in all the organisms and no gross differences were seen among the preparations. PBC III consistently appeared as a doublet on slab gels, which was not observed previously or in the present work when the PBCs were run on tube gels. The lower half of the doublet was very resistant to penicillin, as was previously described (9). The higher molecular weight half of the doublet, on the other hand, was extremely sensitive to all penicillins tested in all of the mutants and the wild type. Kinetics of Cloxacillin Binding to PBCs I, II, and IV. Since radioactive cloxacillin was not available, cloxacillin binding was measured indirectly by competition for subsequent binding of labeled penicillin G (Fig. 4). The concentrations of cloxacillin (,gg/ml) used multiplied times the time of exposure to the antibiotic in minutes range from 0 to 500 in the experiment illustrated. PBC I, totally absent in mutant 5, was no more resistant to cloxacillin in mutant 4 than in the wild type. PBC IV was unaltered in its cloxacillin sensitivity in all of the mutants. PBC II was markedly increased in cloxacillin resistance in both mutants 4 and 5. Quantitative results from a number of such experiments are found in Fig. 5. The concentrations of cloxacillin (,ug/ml X min) required to reduce ['4C]penicillin G binding to each of the PBCs by 50% were estimated from these curves (Table 2). PBC I was unaltered in cloxacillin-resistant mutants 1-4. Its loss or its inability to bind penicillins, therefore, occurred in a single step. PBC II, on the other hand, appeared to undergo three changes in its resistance to cloxacillin, i.e., at mutants 1, 4, and 5. These changes were roughly similar to the changes in the resistance
Mutant 4 5 50 500
500
I
0
1
Mutant 5 5 50 500
Clox.
!
I_-
I
.
II III
IV -
Tq;
FIG. 4. Fluorograms of PBCs prebound with cloxacillin and labeled with [14C]penicillin G. Concentrations of cloxacillin are expressed as wg/ml [14C]penicillin G, except a slight amount by PBC V.
X min. Boiled controls bound no detectable
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Buchanan and Strominger
1819
14 XID
PBC31PBCIZ
PBC I
A .8
AT\ Uv.5 ~~~~~0o '0
~" 3 4
.6
,wr
A
~~~~
2
0
0 80~~~~~~~.
A
500 0 505 10 .5 1 2 -~~~~~~~nwr
0
5
WI
0
c~.2
0
3
A
X..
p~nu 0 .3I1z
-
uSUiV
I1
.i;,
5
5 I 1*v %0
2r-
10-
pAg/ml X min Cloxacillin FIG. 5. Kinetics of cloxacillin binding to the isolated PBCs of B. subtilis wild type and five cloxacillin-resistant mutants. Counts per minute ranged from 300 to 500 for each PBC when no cloxacillin was prebound. The points in the figure are the average of duplicate samples done on the same PBC preparation. In addition, each binding assay was performed on at least two different preparations of PBCs for each organism. These results were not averaged, but were plotted as separate points.
levels of the organisms (Fig. 1). PBC IV retained the same sensitivity to cloxacillin as the wild type in all five mutants. Preliminary measurements with oxacillin and dicloxacillin suggested a similar pattern of changes (data not shown). The affinity of the wild-type and mutant 5 PBCs for penicillin G was also measured. There was no significant change in the sensitivity of either PBC II or PBC IV from the mutant (Fig. 6). DISCUSSION It is clear from these studies that the selection of penicillinresistant mutants of Bacillus subtilis is accompanied by alterations in the penicillin-binding components of this organism. The change in sensitivity of PBC II to cloxacillin appeared to occur in three discrete steps, i.e., in mutants 1, 4, and 5. In mutant 4, and possibly in mutant 1, the change was accompanied by a change in electrophoretic mobility, while in mutant 5 the altered resistance was manifested only by an altered sensitivity to cloxacillin. Earlier a single-step mutant of B. subtilis showing a small change in resistance to 6-APA was isolated; in that mutant also, only PBC II exhibited a significant, reproducible decrease in its affinity for 6-APA (22). Direct proof of the relationship between the altered penicillin-binding component and altered penicillin sensitivity could be obtained by isolating a revertant in which both properties were reverted;
however, it is not possible to select for such a revertant. Alternatively, this problem might be resolved by genetic experiments involving transformation of B. subtilis. There was no apparent change in PBC II or any other PBC in mutants 2 and 3. Some other change must have occurred in these mutants, possibly a membrane change reflected in altered permeability rather than in an alteration in one of the PBCs. Increased production of a cell-bound penicillinase in any of the mutants was ruled out (data not shown). A remarkable fact is that the alteration in sensitivity of mutant 5 to cloxacillin was accompanied by little or no change in the organism's sensitivity to penicillin G. Correspondingly, there was little or no change in the sensitivity of PBC II to penicillin G despite the obvious alteration of the protein's electrophoretic mobility. This indicates that the active sites of penicillin-sensitive proteins may be modified in their sensitivity to one 0lactam antibiotic without any alteration in sensitivity to others. This fact may have implications for therapy with fl-lactam antibiotics and it is paralleled by wide differences in the sensitivity of PBCs in wild-type organisms to different f3-lactam antibiotics. A striking example of this is the extreme sensitivity of PBC II in E. coli to formamidino penicillin as compared to all other penicillins that have been examined (7). The loss of the penicillin-binding activity of PBC I that occurred during isolation of mutant 5 is an interesting finding.
PBC IV
PBC II 1.0
X 1.04 0 m .9
.96
CD .8 c = .7 O
.5
1
.4
Co
.7
.6 6
o-
.6 -
.3 '
Mutant 5
o .2
LL. I
Mutant 5
.5-
.4
U.3 \~~~~~~~~.
.
0 L.
.
-
Wild Type
'lI
0\
M\
.1
)ug/ml
x
.5
i
2
5
min penicillin G
FIG. 6. Kinetics of penicillin G binding to PBCs II and IV of the wild type and mutant 5.
1820
Biochemistry: Buchanan and Strominger
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 2. Cloxacillin resistance of PBCs Mutant
PBC I PBC II PBC IV
Wild type
1
2
3
4
5
0.54 1.75 0.8
1.08 3.8 0.92
1.55 2.65 1.62
1.04 2.18 1.18
0.66 11.4 0.76
72. 1.43
Resistance is defined by the amount of cloxacillin (Atg/ml x min) required to inhibit [14C]penicillin G binding by 50%. These values are read directly from the curves in Fig. 5.
This PBC could not be detected at all by the two techniques that are presently available (isolation of PBCs by affinity chromatography, or their labeling within the membranes with ['4C]penicillin G). However, it is conceivable that PBC I was present'in this mutant, but sufficiently altered in its resistance to penicillins that it escaped detection. When wild-type PBC I is purified, antibody to this protein might be prepared and crossreacting antigenic material sought. Remarkably, mutant 5 grows normally and, even under conditions of stress (growth in minimal media and growth at 470), no abnormality could be detected. The organism also sporulates and germinates normally. If this protein is actually absent in the mutant, then it appears to be unessential to its growth. Previous studies have indicated that PBC V is not essential for growth of B. subtilis (11). Conceivably the fine structure of the cell wall of mutant 5 might be altered by the absence of PBC I, but, in the case of elimination of PBC V from the wild type (by treatment with 6-APA), no alteration in cell wall structure could be found (23). One other possibility is that PBC I, which has the highest molecular weight of the five PBCs of B. subtilis, might be a precursor of one of the other PBCs, and that its absence in mutant 5 might simply be an acceleration of the rate of conversion; however, no evidence for this possibility has been obtained. A relationship between PBC I and PBC II might be suggested by the simultaneous occurrence in mutant 5 of an alteration in the resistance of PBC II and the disappearance of PBC I, since the isolation of a double mutant in a single step is improbable. In any case, PBC I is not likely to be a penicillin killing site, since mutant 5 is still quite sensitive to 6-APA and penicillin G, which have been shown not to bind to PBC I. In addition, PBC IV cannot be the lethal target of cloxacillin, at least, since it did not change in its affinity for cloxacillin in any of the five cloxacillin-resistant mutants. As discussed above, PBC I and PBC V are also eliminated. One of the proteins of the PBC III doublet is too insensitive to be the killing site, and the other is far too sensitive. Thus, all the evidence points to PBC
II as the likely target for killing of B. subtilis by f3-lactam antibiotics. The function of the various penicillin-binding components of B. subtilis remains an important unsolved problem. It may be imagined that these proteins are all enzymes that may participate in different aspects of cell wall synthesis, such as elongation, septation, and formation of "corners" of rods. Further study of mutants in which these PBCs are altered may shed light on these functions, and, certainly, isolation of the individual PBCs in a pure form is an important objective. The work was supported by research grants from the National Institutes of Health (AI-09152) and National Science Foundation (BMS71-01120). C.E.B. was a postdoctoral fellow of the American Cancer Society. 1. Tipper, D. J. & Strominger, J. L. (1965) Proc. Natl. Acad. Sci. USA 54, 1133-1141. 2. Wise, E. M. & Park. J. J. (1965) Proc. Nat!. Acad.- Sci. USA 54, 75-81. 3. Rowley, D., Cooper, P. D., Roberts, P. W. & Smith, E. L. (1950) Biochem. J. 46, 157-161. 4. Eagle, H. (1954) J. Exp. Med.- 99,207-226. 5. Edwards, J. R. & Park, J. T. (1969) J. Bacteriol. 99, 459-462. 6. Suginaka, H., Blumberg, P. M. & Strominger, J. L. (1972) J. Biol.
Chem. 247,5279-5288. 7. Spratt, B. G. & Pardee, A. B. (1975) Nature 254,516-517. 8. Blumberg, P. M. & Strominger, J. L. (1972) Proc. Natl. Acad. Sci. USA 69,3751-3755. 9. Blumberg, P. M. & Strominger, J. L. (1972) J. Brio. Chem. 247, 8107-8113. 10. Blumberg, P. M. & Strominger, J. L. (1974) in Methods in Enzymology, eds. Jakoby,W. B. & Wilchek, M. (Academic Press, New York), Vol. 34, pp. 401-405. 11. Blumberg, P. M. & Strominger, J. L. (1971) Proc. Natl. Acad. Sd. USA 68,2814-2817. 12. Hamilton, T. E. & Lawrence, P. J. (1975) J. Biol. Chem. 250, 6578-6585. 13. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244,4406-4412. 14. Laemmli, U. K. (1970) Nature 227,680-685. 15. Studier, F. W. (1973) J. Mol. Biol. 79,237-248. 16. Vesterberg, O. (1971) Biochim. Biophys. Acta 243,345-348. 17. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46,83-88. 18. Demerec, M. (1948) J. Bacteriol. 56,63-74. 19. O'Callaghan, C. H. & Muggleton, P. W. (1972) Cephalosporins and Penicillins. Chemistry and Biology, ed. Flynn, E. H. (Academic Press, New York), pp. 438-495. 20. Linn, T., Losick, R. & Sonenshein, A. L. (1975) J. Bacteriol. 122, 1387-1390. 21. Swaney, J. B., Vande Woulde, G. F. & Bachrach, H. L. (1974)
Anal. Biochem. 58,337-346.
22. Blumberg, P. M. (1973) Ph.D. Dissertation, Harvard University. 23. Sharpe, A., Blumberg, P. M. & Strominger, J. L. (1974) J. Bac-
teriol. 117,926-927.
