Proc. Nati. Acad. Sci. USAVol. 73, No. 6, pp. 1816-1820, June 1976Biochemistry

Altered penicillin-binding components in penicillin-resistantmutants of Bacillus subtilis

(fluorography/affinity chromatography)

CHRISTINE E. BUCHANAN AND JACK L. STROMINGERThe Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

Contributed by Jack L. Strominger, January 26, 1976

ABSTRACT Penicillin- (cloxacillin-) resistant mutants ofBacillus subtilis were isolated in a stepwise fashion and the fivepenicillin-binding components (PBCs) in each were examinedto determine which of the proteins, if any, corresponds to thepenicillin killing site. PBCs III and V were previously elimi-nated as the likely penicillin target. In the present work, PBCIV showed no change in sensitivity to cloxacillin in any of theresistant mutants isolated. PBC I did not change until thefifth-step mutant, in which it could not be detected by penicillinbinding. Since PBC I did not bind penicillins that are lethal forthis mutant, it also cannot be the lethal target. PBC II showedincreased resistance to cloxaciffin in three discrete steps, i.e.,in mutants 1, 4, and 5, accompanied by changes in its electro-phoretic mobility. However, the sensitivity of PBC II to peni-cillin C changed very little. Correspondingly, the cloxacillin-resistant mutants were unaltered in their sensitivity to penicillinG in vivo. Thus, of the five PBCs found in B. subtifis, PBC II isthe most likely target for killing by penicillins.

Penicillin kills bacteria by inhibiting the crosslinking of thebacterial cell wall catalyzed by a transpeptidase (1, 2). Theantibiotic is believed to be a substrate analogue of the enzymeand to inactivate the transpeptidase by forming a stable peni-cilloyl-enzyme intermediate (1). In fact, a similarity has beenfound in the concentration of penicillin required to kill bacteriaand to saturate their penicillin killing sites (3-5). The inter-pretation has recently been complicated, however, by thedemonstration of multiple penicillin-binding proteins in suchorganisms as Escherichia coli, Staphylococcus aureus, Bacillussubtilis, Bacillus stearothermophilus, and Bacillus cereus (6,7). Not all of these proteins that specifically bind penicillincorrespond to the lethal target of penicillin, nor does any oneprotein necessarily have the same sensitivity to each of a varietyof 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 ap-proach to this problem is to determine which components be-come more resistant to penicillin upon mutation of the organismto greater penicillin resistance.

Bacillus subtilis has five penicillin-binding componentswhich can be easily isolated from solubilized membranes byaffinity chromatography (8-10). Of the five, only PBC V hasso far been found to possess enzymatic activity. It is the D-ala-nine carboxypeptidase and represents at least 80% of the pen-icillin-binding protein both in the membranes and in the pu-rified PBCs (8, 11). Since this enzyme is not essential to normalgrowth in B. subtilis it is not a penicillin killing site (11). Inaddition, PBC III is not a killing site because it binds penicillinonly at very high concentrations (9). On the other hand, PBCsI, II, and IV are all candidates for the penicillin killing site, sincetheir antibiotic sensitivity profiles closely resemble that of the

killing site (9). Thus, the present study has concentrated onchanges that might occur in PBCs I, II, and IV in penicillin-resistant mutants of B. subtilis.

MATERIALS AND METHODSOrganism and Isolation of Mutants. B. subtilis strain Por-

ton, resistant to 5 ,ug/ml of rifampicin and 200 /.g/ml ofstreptomycin, was used as the wild type. Penicillin sensitivitywas determined by counting colonies on Antibiotic Medium3 (Difco) agar plates containing various concentrations of theantibiotic at 30. To isolate a resistant mutant, a colony growingon a plate containing a high concentration of cloxacillin (E. R.Squibb & Sons, Inc.) was picked and purified by two rounds ofsingle colony isolation. No mutagens were used. Cell-boundpenicillinase activity was assayed as described (12).

Preparation of Membranes and Isolation of PBCs. Mem-branes were prepared by grinding cells with glass beads as de-scribed previously (9). The penicillin-binding components wereisolated by affinity chromatography as described (10) exceptthe Sepharose 4B200 (Sigma) was coupled with 6-aminohexa-noic acid (Aldrich) prior to 6-aminopenicillanic acid (6-APA)(Sigma) substitution.

Penicillin Binding Assays. [14C]Penicillin G (Amersham-Searle, 53 mCi/mmol) was bound to B. subtilis membranes asdescribed (9). Sensitivity of the purified PBCs to a variety ofpenicillins was measured by first prebinding with an unlabeledpenicillin at different concentrations and then adding a satu-rating concentration of [14C]penicillin G. The protocol wassimilar to that described elsewhere (8). The binding reactionwas stopped by adding a 250-fold excess of unlabeled penicillinG, followed by precipitation of the protein with 80% acetone.The protein precipitate was dried with a stream of air, boiledin sample buffer (see below), and applied to a gel.Sodium Dodecyl Sulfate (NaDodSO4) Polyacrylamide Gel

Electrophoresis. Tube gels were run as described by Weberand Osborn (13). The gels were 8 cm long and subjected toelectrophoresis for 6 hr at 8 mA per gel. Discontinuous Na-DodSO4 polyacrylamide slab gels (2 mm) were run as described(14, 15). The upper stacking gel was 3% acrylamide and therunning gel was 7.5% acrylamide. The sample buffer contained10% (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 5min, were subjected to electrophoresis at 110 V for 3.5 hr. Thegel was stained with Coomassie brilliant blue (16) for 2 hr at 370followed by overnight destaining with 5% methanol and 7.5%acetic acid. "C-Labeled proteins in the dried slab gel weredetected by fluorography (17). To quantitate the results, thex-ray film (after 1-4 days' exposure) was scanned with a dou-ble-beam recording microdensitometer manufactured by Joyce,Loebl and Co. Ltd.

1816

Abbreviations: PBC, penicillin-binding component; 6-APA, 6-ami-nopenicillanic acid; NaDodSO4, sodium dodecyl sulfate.

Table 1. Sensitivity of wild type and mutant 5 to various penicillins

LD50 (gg/ml, 30°)

Penicillin Structure WT Mutant 5

NH,, S CH36-Aminopenicillanic acid CjCH3 7.0 11.0

COOHO H

Benzylpenicillin (penicillin G) JCHC-N S OH3 0005 0.012N CH,3005OHN COOH

Cloxacillin CH N S CH30.1 18.0

NcKi OH3 N OH3OxacCOON>CQOH

OHIDicloxacillin

C-N S OH3 00 .

c IN t..cH N OH3 00 .O POOH

Oxacillin C-N S OH3 0.09 3.5N OCH.3 N H3

O O~~OOH

RESULTSIsolation of a Highly Penicillin Resistant Mutant. High-

level penicillin resistance in bacteria develops in a stepwisefashion in the absence of penicillinase production (18). A sin-gle-step penicillin-resistant mutant shows only a small increasein penicillin resistance. Since small changes in the binding ofpenicillin 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 isolatedby a series of stepwise increases in resistance. Cloxacillin waschosen as the selective penicillin because it is relatively resistant

10l4r

10-6

.05 .1 .5 1.0 5.0 l0 40

,ug/ml Cloxacillin

FIG. 1. Survival curves of B. subtilis and the five mutants. Thecurves (from left to right) correspond to survival of the wild type,mutants 1, 2, 3, 4, and 5. The arrows indicate where a survivor waspicked to be used for the isolation of the next step mutant. The plateswere incubated at 30° to avoid selecting against any temperature-sensitive mutants that might occur. The LDWos (jg/ml), defined as

the concentrations of cloxacillin required to reduce the number ofcolony-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.

to the fl-lactamase of Bacillus species (19). The survival curvesof the wild-type B. subtilis and five cloxacillin-resistant mutants(Fig. 1) illustrate the relatively small increase in cloxacillinresistance occurring in each step of the selection.

Sensitivity of Mutant 5 to Other Penicillins. The mutantshowed greatly increased resistance to oxacillin (Squibb) anddicloxacillin (Bristol Laboratories) as well as to cloxacillin (Table1). Only a slight change in sensitivity to 6-APA or penicillin Gwas found.

Wild Mutant No.Type 1 2 3 4 5

II,

*

FIG. 2 NaDodSO4 polyacrylamide gel ofPBCs isolated by affinitychromatography from B. subtilis and five cloxacillin-resistant mu-tants. The protein above PBC Tin each sample is apparently unre-duced penicillin-binding protein. In the absence of mercaptoethanolthere was even a greater amount of protein in this area of the gel.Visible bands, other than the five PBCs, were not reproducible.

Proc. Natl. Acad. Sci. USA 73 (1976) 1817Biochemistry: Buchanan and Strominger

1818 Biochemistry: Buchanan and Strominger

A B C

-I

- 11

- III-IV

-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 PBCpreparations. The altered mobility of PBC II from mutant 5 was ev-ident in at least six different preparations.

Isolation of PBCs by Affinity Chromatography. Two in-teresting alterations were evident in stained NaDodSO4 gels ofthe PBCs purified by affinity chromatography (Fig. 2). Inmutant 5, PBC I was not present. Either it did not bind to the6-APA of the affinity column, e.g., because it was insensitiveto penicillin, or the protein was absent in this mutant. The latterinterpretation is supported by the observation that when[14C]penicillin G at high concentration was bound to mem-branes of mutant 5, no radioactive PBC I could be found, evenin trace amount.A more subtle difference was found in the protein pattern

of PBC II. PBC II appeared to have a slightly slower mobilityin some of the mutants than in the wild type. An examinationof PBC II in the series of mutants (Fig. 2) suggests there wereseveral discrete changes in the mobility of PBC II, one evident

in mutants 4 and 5 as compared to mutant 3. In order to es-tablish this change more firmly, the mutant 5 and wild-typesamples were run separately in adjacent wells and together ina mixture on a slab gel (Fig. 3). The pattern clearly indicatesthat PBC II of mutant 5 is not identical to that of the wild type.Another slight alteration in PBC II may have occurred in mu-tant 1. Such small changes in electrophoretic mobility could bethe result of a single amino acid change affecting the chargeon the protein (20, 21). Examination of PBC II is further com-plicated by the fact that in some gels it appears to be two pro-teins. Further studies using higher resolution gel systems arenecessary.PBCs III, IV, and V were present in all the organisms and no

gross differences were seen among the preparations. PBC IIIconsistently appeared as a doublet on slab gels, which was notobserved previously or in the present work when the PBCs wererun on tube gels. The lower half of the doublet was very resistantto penicillin, as was previously described (9). The higher mo-lecular weight half of the doublet, on the other hand, was ex-tremely sensitive to all penicillins tested in all of the mutantsand the wild type.

Kinetics of Cloxacillin Binding to PBCs I, II, and IV. Sinceradioactive cloxacillin was not available, cloxacillin binding wasmeasured indirectly by competition for subsequent binding oflabeled penicillin G (Fig. 4). The concentrations of cloxacillin(,gg/ml) used multiplied times the time of exposure to the an-tibiotic in minutes range from 0 to 500 in the experiment il-lustrated. PBC I, totally absent in mutant 5, was no more re-sistant to cloxacillin in mutant 4 than in the wild type. PBC IVwas unaltered in its cloxacillin sensitivity in all of the mutants.PBC II was markedly increased in cloxacillin resistance in bothmutants 4 and 5.

Quantitative results from a number of such experiments arefound in Fig. 5. The concentrations of cloxacillin (,ug/ml X min)required to reduce ['4C]penicillin G binding to each of the PBCsby 50% were estimated from these curves (Table 2). PBC I wasunaltered in cloxacillin-resistant mutants 1-4. Its loss or its in-ability to bind penicillins, therefore, occurred in a single step.PBC II, on the other hand, appeared to undergo three changesin its resistance to cloxacillin, i.e., at mutants 1, 4, and 5. Thesechanges were roughly similar to the changes in the resistance

Wild Type0 1 5 50 500I I I I

Mutant 45 50 500

!

Mutant 50 1 5 50 500 Clox.

I .

FIG. 4. Fluorograms of PBCs prebound with cloxacillin and labeled with [14C]penicillin G. Concentrations of cloxacillin are expressed as wg/mlX min. Boiled controls bound no detectable [14C]penicillin G, except a slight amount by PBC V.

IIIII -

IV -

WOW.

Proc. Natl. Acad. Sci. USA 73 (1976)

I_-

Tq;

Proc. Natl. Acad. Sci. USA 73 (1976) 1819

XID

.8

.6

A

c~.2

0 .5 1 2 5 10

pAg/ml X min CloxacillinFIG. 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 onthe 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 samesensitivity to cloxacillin as the wild type in all five mutants.Preliminary measurements with oxacillin and dicloxacillinsuggested a similar pattern of changes (data not shown).The affinity of the wild-type and mutant 5 PBCs for peni-

cillin G was also measured. There was no significant change inthe sensitivity of either PBC II or PBC IV from the mutant (Fig.6).

DISCUSSIONIt is clear from these studies that the selection of penicillin-resistant mutants of Bacillus subtilis is accompanied by alter-ations in the penicillin-binding components of this organism.The change in sensitivity of PBC II to cloxacillin appeared tooccur in three discrete steps, i.e., in mutants 1, 4, and 5. Inmutant 4, and possibly in mutant 1, the change was accompa-nied by a change in electrophoretic mobility, while in mutant5 the altered resistance was manifested only by an alteredsensitivity to cloxacillin. Earlier a single-step mutant of B.subtilis showing a small change in resistance to 6-APA wasisolated; in that mutant also, only PBC II exhibited a significant,reproducible decrease in its affinity for 6-APA (22). Direct proofof the relationship between the altered penicillin-bindingcomponent and altered penicillin sensitivity could be obtainedby isolating a revertant in which both properties were reverted;

however, it is not possible to select for such a revertant. Alter-natively, this problem might be resolved by genetic experimentsinvolving transformation of B. subtilis.

There was no apparent change in PBC II or any other PBCin mutants 2 and 3. Some other change must have occurred inthese mutants, possibly a membrane change reflected in alteredpermeability rather than in an alteration in one of the PBCs.Increased production of a cell-bound penicillinase in any of themutants was ruled out (data not shown).A remarkable fact is that the alteration in sensitivity of mu-

tant 5 to cloxacillin was accompanied by little or no change inthe organism's sensitivity to penicillin G. Correspondingly, therewas little or no change in the sensitivity of PBC II to penicillinG despite the obvious alteration of the protein's electrophoreticmobility. This indicates that the active sites of penicillin-sen-sitive proteins may be modified in their sensitivity to one 0-lactam antibiotic without any alteration in sensitivity to others.This fact may have implications for therapy with fl-lactamantibiotics and it is paralleled by wide differences in the sen-sitivity of PBCs in wild-type organisms to different f3-lactamantibiotics. A striking example of this is the extreme sensitivityof PBC II in E. coli to formamidino penicillin as compared toall other penicillins that have been examined (7).The loss of the penicillin-binding activity of PBC I that oc-

curred during isolation of mutant 5 is an interesting finding.

PBC II1.0

.96

.7

.6

Mutant 5 .5-

.4

-U.3 -

.\~~~~~~~~.

0\ 'lI

.

PBC IV

Mutant 5

Wild Type

.1 .5 i 2 5

)ug/ml x min penicillin GFIG. 6. Kinetics of penicillin G binding to PBCs II and IV of the wild type and mutant 5.

14

A PBCI PBC31PBCIZ

~" 3 AT\4 ~~~~~0o'0 Uv.5 0 5

,wr ~~~~ 2 0 3

0 WI80

A 0~~~~~~~. X.. -

-~~~~~~~nwr A- p~n I1 2 I5 1 5

X 1.04

0m .9

CD .8c= .7

O6.6

o- .5 -

1 .4

Co .3 '

o .20L.LL. I

Biochemistry: Buchanan and Strominger

10-50 500 0 .3I1z0 u uSUiV .i;, r- *v%0

M\

1820 Biochemistry: Buchanan and Strominger

Table 2. Cloxacillin resistance of PBCs

MutantWildtype 1 2 3 4 5

PBC I 0.54 1.08 1.55 1.04 0.66PBC II 1.75 3.8 2.65 2.18 11.4 72.PBC IV 0.8 0.92 1.62 1.18 0.76 1.43

Resistance is defined by the amount of cloxacillin (Atg/ml x min)required to inhibit [14C]penicillin G binding by 50%. These valuesare read directly from the curves in Fig. 5.

This PBC could not be detected at all by the two techniques thatare presently available (isolation of PBCs by affinity chroma-tography, or their labeling within the membranes with['4C]penicillin G). However, it is conceivable that PBC I waspresent'in this mutant, but sufficiently altered in its resistanceto penicillins that it escaped detection. When wild-type PBCI is purified, antibody to this protein might be prepared andcrossreacting antigenic material sought. Remarkably, mutant5 grows normally and, even under conditions of stress (growthin minimal media and growth at 470), no abnormality couldbe detected. The organism also sporulates and germinatesnormally. If this protein is actually absent in the mutant, thenit appears to be unessential to its growth. Previous studies haveindicated that PBC V is not essential for growth of B. subtilis(11). Conceivably the fine structure of the cell wall of mutant5 might be altered by the absence of PBC I, but, in the case ofelimination of PBC V from the wild type (by treatment with6-APA), no alteration in cell wall structure could be found (23).One other possibility is that PBC I, which has the highest mo-lecular weight of the five PBCs of B. subtilis, might be a pre-cursor of one of the other PBCs, and that its absence in mutant5 might simply be an acceleration of the rate of conversion;however, no evidence for this possibility has been obtained. Arelationship between PBC I and PBC II might be suggested bythe simultaneous occurrence in mutant 5 of an alteration in theresistance of PBC II and the disappearance of PBC I, since theisolation of a double mutant in a single step is improbable. Inany case, PBC I is not likely to be a penicillin killing site, sincemutant 5 is still quite sensitive to 6-APA and penicillin G, whichhave 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 inany of the five cloxacillin-resistant mutants. As discussed above,PBC I and PBC V are also eliminated. One of the proteins ofthe PBC III doublet is too insensitive to be the killing site, andthe 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 an-tibiotics.The function of the various penicillin-binding components

of B. subtilis remains an important unsolved problem. It maybe imagined that these proteins are all enzymes that may par-ticipate in different aspects of cell wall synthesis, such as elon-gation, septation, and formation of "corners" of rods. Furtherstudy of mutants in which these PBCs are altered may shed lighton these functions, and, certainly, isolation of the individualPBCs in a pure form is an important objective.

The work was supported by research grants from the National In-stitutes of Health (AI-09152) and National Science Foundation(BMS71-01120). C.E.B. was a postdoctoral fellow of the AmericanCancer Society.

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Proc. Natl. Acad. Sci. USA 73 (1976)