Vol. 140, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 955-9630021-9193/79/12-0955/09$02.00/0

Escherichia coli Mutants Tolerant to Beta-LactamAntibiotics

KAZUAKI KITANO AND ALEXANDER TOMASZ*The Rockefeller University, New York, New York 10021

Received for publication 20 July 1979

Two types of Escherichia coli mutants tolerant to beta-lactam antibiotics wereisolated. One is E. coli X2452, which showed a tolerant response against beta-lactam antibiotics when grown at 420C, and the others are the mutants C-80 andC-254, selected from mutagenized E. coli X 1776 by cycles ofexposure to ampicillin,cephaloridine, and starvation of the nutritionally required diaminopimelic acid.Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of X1776or in X2452 grown at 320C. In contrast, the same antibiotics caused only areversible inhibition of growth in mutants C-80 and C-254 or in cultures of X2452grown at 420C. Beta-lactam antibiotics that show high affinity for penicillin-binding proteins 2 or 3 (mecilhinam and cephalexin, respectively) induced similarmorphological effects (ovoid cell formation and filament formation) in both parentand mutant strains. In contrast, beta-lactam antibiotics which have a high affinityfor penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapidlysis in the parental strains, caused cell elongation in the tolerant bacteria. Incontrast to the parental cells, autolytic cell wall degradation was not triggered bybeta-lactam treatment of X2452 cells grown at 420C or in mutants C-80 and C-254. The total autolytic activity of mutants C-80 and C-254 was less than 30%that of the parent strain. However, virtually identical autolytic activities werefound in cells of X2452 grown either at 42 or 320C. Possible mechanisms for thepenicillin tolerance of E. coli are considered on the basis of these findings.

Several types ofbiochemical observations sug-gest that lysis ofEscherichia coli by beta-lactamantibiotics involves the activity of autolytic en-zymes (5, 18, 19). Evidence for such an involve-ment exists in pneumococci since mutants se-lected for a defective autolytic system werefound to be resistant to the lytic (and, to somedegree, to the bactericidal) effect of beta-lactamantibiotics, while remaining sensitive to thegrowth inhibitory effect of these antibiotics (25,26). Repeated attempts to select for autolysin-defective E. coli have failed so far. Therefore,we decided to select for penicillin-tolerant mu-tants of E. coli, i.e., bacteria that could survivetreatment with penicillin in a manner that ischaracteristic of the penicillin-tolerant pneu-mococci. We describe here three tolerant E. coliisolates. Each mutant was found to have a de-fective autolytic system.We chose the E. coli K-12 strain X1776 as the

parental strain in the mutant isolation becauseof the presence of several useful properties ofwhich ofparticular importance were the require-ment for diaminopimelic acid (DAP), defectiveouter membrane, and sensitivity to detergent-induced lysis (4).

Although a complete characterization of thegenetic and biochemical basis of tolerance inthese bacteria is not yet available, it was feltthat the existence of the first penicillin-tolerant,gram-negative mutants was of sufficient interestto warrant a preliminary description.

MATERIALS AND MErTODSBacterial strains and culture conditions. E. coli

X2452 F- dapD AlacZ39 A(galbchl) A- tyrT58 naLA29AthyA57 IyaA32 endAl asd hsdS3 (4), E. coli X1776F- tonA53 dapD8 minAl supE42 A(gal-uvrB)40 A-minB2 rfb-2 nalA25 oms-2 thyA57 metC65 oms-1A(bioH-asd)29 cycB2 cycAl hsdR2, and E. coli C-80and C-254 (beta-lactam tolerant mutants derived formX1776) were used. Because of the known mechanicalfragility of both strains X1776 and x2452, bacteria werecultured without aeration. Cultures of 10 ml each weregrown in test tubes (18 by 150 mm) at 32 or 420C inPenassay broth (Difco antibiotic medium no. 3) sup-plemented with DAP (20 ,ug/ml), L-lysine (100 yig/ml),biotin (0.2 ,ug/ml), and thymidine (30,ug/ml) (completemedium, OM). Cells radioactively labeled in the cellwall with DAP were obtained by growth in medium inwhich the DAP was replaced with 1 uCi of [3H]DAPper ml (giving a final DAP concentration of 3.1 ,ug/ml).

Isolation of mutants. E. coli X1776 was mutagen-955

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956 KITANO AND TOMASZ

ized by conventional procedures (1). Bacteria, grownexponentially at 320C in complete medium, were sus-pended in 0.05 M Tris-maleate buffer (pH 6.0) con-taining 300 ,ug of N-methyl-N'-nitro-N-nitrosoguani-dine and were incubated for 30 min at 37°C. Celis werewashed once with Tris-maleate buffer to remove themutagen and suspended in OM. Portions of 2 ml wereinoculated into 8 ml of OM and incubated at 320C for3.5 h (cell concentration at this point was 5 x 107 to 7x 107 cells per ml). Mutants were selected next by acycle of treatments that included the following steps.

Step (i). A 10-ml mutagenized culture was treatedwith ampicillin (20 ug/ml, corresponding to about fivetimes the minimal inhibitory concentration [MIC]) for1.2 h. During this treatment, the turbidity of thebacterial culture first increased by about 30% and thendropped to about 50% of the maximum turbidity value.The cells were collected by centrifugation (5,000 x g,10 min, room temperature), washed once with OM,and resuspended in fresh OM (twice the original vol-ume). After overnight incubation (13 h) at 320C, thesurviving cells grew to a turbid culture that was resus-pended in fresh OM and allowed to double in itsturbidity (5 x 107 to 10 x 107 per ml), at which time a10-ml portion of the culture received step (ii) of theenrichment procedure.

Step (ii). Step (ii) consisted of treatment withcephaloridine (20 jug/ml; five times the MIC value) at320C for 1.5 h. The turbidity changes of this culturewere about the same as those observed during the firstampicillin treatment. After the cells were washed withOM, dilution and overnight incubation were the sameas after ampicillin treatment. The overnight culturewas washed with OM without DAP, resuspended inOM without DAP, and incubated at 320C for 3 h.Step (iii). During this DAP starvation there was an

initial 20% increase followed by a decline (about 30%of the maximum) in turbidity. The culture was washedand resuspended in OM (to half of the culture's tur-bidity at the end ofthe DAP starvation) and incubated(320C) for 3 h, during which time the turbidity dou-bled. Portions of 10 ml of this culture were thenexposed to the final step.Step (iv). Step (iv) consisted of ampicillin treat-

ment (20 ,g/ml for 2.5 h). The bacteria were finallywashed with OM and resuspended in 10 ml of OM,and frozen stocks were prepared (freezing at -40°Cand storage at -80°C).The final scoring of mutants was done in the follow-

ing way: frozen stock cultures were melted, diluted,and plated on OM agar plates (about 107 bacteria perplate) containing 10 Ag of ampicillin per ml and incu-bated at 320C for 16 h. Then the plates were overlay-ered with 4 ml of soft agar medium (0.6% agar in OMmedium) containing 10 U of penicillinase (RikersChemicals) per ml and incubated for another 24 h.The colonies that grew up under this condition (about100 per plate) were picked into liquid medium andchecked for a tolerant response against beta-lactamantibiotics. Out of 340 such surviving colonies, 6 werefound to show a substantially decreased rate of loss ofturbidity (as compared with the parental culture)when challenged with cephaloridine (five times theMIC). These cultures also showed lower autolyticactivity (30 to 60% of the parental cells). Two of these

tolerant mutants, C-80 and C-254, exhibiting virtuallyno lysis during cephaloridine treatment, were chosenfor more detailed characterization to be described inthis paper.Assay procedures. Bacteria were routinely grown

at 320C except when noted otherwise. Culture growthand culture lysis were monitored with a ColemanNepho-colorimeter (14). Viable titers of the cultureswere assayed by routine plating procedures. The assaymethod of triggered autolysin by beta-lactam anti-biotics (13) was as follows.

After growth in medium supplemented with [3H]-DAP for several generations, cells in the exponentialgrowth phase were collected by centrifugation (4,300x g, 5 min), transferred to radioactive isotope-freegrowth medium, and incubated for 50 min (i.e., aperiod of about one generation) to deplete cellularpools of the [3H]DAP. After this period, 1.5-ml por-tions of the culture were distributed into a number ofsmall tubes containing beta-lactam antibiotics at var-ious concentrations (representing multiples of the cor-responding MICs) and incubated for an additional 10to 20 min. The cultures were chilled (ice bath) andthen centrifuged at 3,300 x g for 5 min at 40C. Cellswere washed with 1.5 ml of ice-cold phosphate buffer(0.1 M, pH 7.0), resuspended in 1.5 ml of the samebuffer containing 10 mM MgSO4, and incubated at320C. The total time needed to transfer the cells tothe buffer was about 10 to 15 min. After 0, 30, 60, 90,and 120 min of incubation, 200-p1 portions were re-moved into prechilled Eppendorf microcentrifugetubes containing 20 pl of 38% formaldehyde (to stopmurein hydrolase activity).

After centrifugation at 12,000 x g for 10 min in thecold (40C), radioactivity in 100 pl of the supernatantfluids was counted. To determine total radioactivity ofthe reaction mixture, 200-pd portions were mixed with20 p1 of 4% deoxycholate and incubated for 30 min at320C; a 100-pl portion of this suspension was used todetermine radioactivity. The activity of autolysin trig-gered by beta-lactam antibiotics was expressed as therate of degradation of murein during a 2-h incubationof the beta-lactam antibiotic-treated cells in buffer.The rates were corrected for the spontaneous rate ofrelease of radioactivity from the control (untreated)cells. The treatment time with beta-lactam antibioticswas 20 min (for E. coli X2452 cells) or 10 min (for thecells of E. coli x1776 C-80 and C-254).

Triggering of murein hydrolase by trichloro-acetic acid. The method of Schwarz et al. was used(18) (see also legend to Fig. 8) to trigger mureinhydrolase by trichloroacetic acid.

Reagents. DL-(meso)-2,6-DiamiIno-[U-3H]pimelicacid (1.5 Ci/mmol) was purchased from AmershamCorp. (Arlington Heights, Ill.). All other materials andchemicals were reagent-grade commercially availableproducts.

RESULTSTolerant response ofE. coli X2462 to beta-

lactam antibiotics at 420C. Several mutantsof E. coli K-12 were examined for their responseto beta-lactam antibiotics and a mutant, E. coliX2452, was found to show a tolerant response to

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beta-lactams at 42°C but not at 32°C (Fig. 1).X2452 was isolated by Dennis Pereira in thelaboratory of Roy Curtiss (D. Pereira, Ph.D.Thesis, University of Alabama Medical Center,Birmingham, Ala., 1979). Although rapid culturelysis occurred after exposure to the MIC ofcephaloridine at 320C, the cells were stable tolysis by the addition of cephaloridine when thebacteria were grown at 420C. This phenomenonwas reproducible by the addition of other beta-lactams such as ampicillin, benzylpenicillin, orcephalothin. The MICs of these beta-lactamswere identical at the two temperatures (Table1).The tolerant response to beta-lactam anti-

biotics was temperature dependent, and if tem-perature was shifted down from 42 to 320C at 0to 30 min after the addition of the drug, cellsstarted lysing after a short time lag (Fig. 2).Tolerant response of E. coli C-80 and C-

254 to beta-lactam antibiotics. Figure 3 dem-onstrates the effect of cephaloridine on culturesof E. coli X1776, C-80, and C-254. Althoughgrowth of the mutants was stopped by the ad-dition of cephaloridine, the cells scarcely lysedeven after prolonged (overnight) incubation withthe antibiotic. Culture lysis was very slow ornegligible in all of the tolerant mutants (includ-ing X2452 grown at 4200) even during exposure

0 1 2 3

HOURSFIG. 1. Effect of cephaloridine

coli X2452 growing at 32 and 42°C.were grown in OM without aeraexponential phase of growth (arrreceived cephaloridine at the conc

ples of MIC; 7.8 pg/ml at both tecated by the numbers, and the groubacterial cultures was followed by iplotted in nephelometric (N) units.

42 C

TABLE 1. MICs of beta-lactam antibiotics againstmutants of E. colia

MIC (jug/ml) of strain:

Antibiotic X2452X1776 C-80 C-254

320C 420C

Cephaloridine 3.9 3.9 2.0 3.9 3.9Ampicillin 3.9 3.9 3.9 3.9 3.9Cephalothin 15.6 15.6 31.25 62.5 62.5Benzylpenicillin 31.25 31.25 31.25 62.5 62.5Cefoxitin 7.8 7.8 7.8Cephalexin 15.6Mecillinam 7.8 7.8 7.8

aThe MICs listed in the table were determined by thefollowing procedure: exponentially growing cultures (at cellconcentration ofabout 5 x 107 cells per ml) received antibioticsat various concentrations, and growth of the cultures wasmonitored (as described in the Materials and Methods). Theminimum antibiotic concentration that caused cessation ofgrowth was taken as the MIC. Inhibition of growth was notinstant but became manifest only after a residual increase inthe turbidity of cultures.

400t

N 200}

100'

50'

0 1 2

HOURSFIG. 2. Effect of temperature shift during treat-

ment of E. coli X2452 with cephaloridine. Bacterialcultures were grown in OM at 42°C without aeration.At a cell concentration of about 1 x io0 viable cellsper ml (in the late exponential phase of growth)(arrow), the cultures received 2.5 times the MIC ofcephaloridine (19.5 pg/ml), and the temperature of

0 1 2 3 the cultures was shifted to 320C at 0 (A), 15 (V), and30 (El) min after the addition of the drug. Controlcultures at 42°C with (O) or without (*) cephalori-

on cultures of E. dine are also shown.Bacterial culturesition. In the late to high concentrations (eight times the MIC) ofrow), the cultures cephaloridine, ampicillin, and a variety of other,mperatures) indi beta-lactam antibiotics. Nevertheless, inhibitorspth response of the of early stages of cell wall synthesis (e.g., thenephelometry and combination of D-cycloserine and fluoro-D-ala-

nine) could still induce lysis of the tolerant bac-

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400

N

X-1 776

1004

so

0 1 2 3

C-254

0 1 2 3

C-80

0 1 2 3HOURS

FIG. 3. Effect of cephaloridine on cultures of E. coli X1776 and its mutants C-80 and C-254. Bacterialcultures were grown in OM at 32°C without aeration. In the exponential phase ofgrowth (5 x 107 viable cellsper ml) (arrow), the cultures received antibiotics at the concentrations (multiples of MIC) indicated by thenumbers, and the growth response of the bacterial cultures was followed by nephelometry. The MICs forcephaloridine were as follows: xl776, 3.9 pg/ml; mutants C254 and C-80, 7.8 pg/ml.

teria (data not shown).The MICs of various beta-lactams (for

method of MIC determination see footnote a,Table 1) were identical to those of the parentalcells (Table 1) except for benzylpenicilhin andcephalothin; in these cases the MIC for thetolerant cells was higher (maximum, twice thoseof the parents).

Cultivation of E. coli X2452 at 420C gave astriking protection against the killing effects ofcephaloridine (Fig. 4a) and also against lysis.The killing effects of cephaloridine in the mu-tants C-80 and C-254 were also very weak ascompared with the effects in the parental strain(Fig. 4b).Morphological changes of the mutant

cells during treatment with beta-lactams.Figure 5 demonstrates the effect of three beta-lactam antibiotics on the cell shape of E. coliX2452 at 42 and 320C. Cephaloridine, which hasa high affinity for penicillin-binding protein(PBP) lb (20, 21) caused rapid lysis of cells at320C and the formation of empty cells, whereasat the nonpermissive temperature for lysis, cellelongation continued, and bacilli became notice-ably longer than the control cells. Mecillinam(which has a high affinity for PBP 2 [20]) causedthe formation of ovoid cells at both 32 and 420C.During the 3 h of treatment with cephalexin,cells grown at 420C have elongated to lengthsvarying between 1.5 and 3 times the length ofthe untreated bacteria grown at this tempera-ture. The same drug caused a more substantialelongation (3 to 6 times the normal length) when

x

4._co

2

CX-254

C°~X-1776B

1 2

HOURSFIG. 4. Bactericidal action of cephaloridine

against beta-lactam-tolerant mutants of E. coli. (A)E. coli X2452 cells growing in complete medium at 32and 42°C were treated at 0 min (cell concentration, 1x 108 bacteria per ml) with 20 pg (about 2.5 times theMIC) of cephaloridine per ml. (B) Cells of E. coliX1776 C-80 and C-254 growing in complete mediumat 320C were treated at 0 min with twice the MIC ofcephaloridine. Samples were periodically removed,diluted with complete medium, and plated to deter-mine the number of viable cells.

the cells were grown at 320C. Similar phenom-ena were also observed with the tolerant mu-tants C-80 (Fig. 6) and C-254 (data not shown).In contrast, the mecillinam-induced ovoid cellformation was not easy to document in thesetolerant strains or in the parental strain X1776.Mecillinam treatment, at least under the condi-tions used here (i.e., relatively high cell concen-

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-4

........ ....t~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.....~~* .j .. .t *$,. b

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FIG. 5. Effect of beta-lactam antibiotics on cell shape of E. coli X2452 grown at 32 and 42°C. (A) Control;(B) cephaloridine; (C) mecillinam; (D) cephalexin. Cells grown at 32 and 42°C were exposed to four times theMICs of various beta-lactam antibiotics for 3 h. Cells were fixed with 2% glutaraldehyde, andphase-contrastmicrographs were then made ofthe cells, using a Zeiss microscope fitted with a Planachromat 100/1.25phase-contrast oil immersion objective. Kodak Panatomic-X film was used for the photomicrography.

tration), caused the formation of abnormallyshaped bacteria (Fig. 6).Triggering of autolytic activity by ceph-

aloridine and by trichloroacetic acid treat-ment. The autolytic cell wall degradation of E.coli X2452 was effectively triggered by brief ex-posure (10 to 20 min) to beta-lactam antibioticsat the lysis-permissive temperature but was nottriggered at 42°C (Fig. 7). Autolysis of mutantsC-80 and C-254 was not triggered by this treat-ment either (Fig. 7). Interestingly, exposure tohigh concentrations of antibiotics (eight timesthe MIC [Fig. 7]) has occasionally caused asuppression of the rate of cell wall degradation.This type of effect has been consistently ob-served at very high concentrations (10 to 100times the MICs) of certain beta-lactam antibiot-ics (13).The autolytic activity of E. coli X2452 trig-

gered by 5% trichloroacetic acid treatment (7)was almost the same in cells grown either at 32or 420C and was independent of the temperatureat which the assays were carried out (Fig. 8a).In contrast, the autolytic activities of C-80 andC-254 triggered by 5% trichloroacetic acid treat-ment were less than 30% that ofthe parent strain

(Fig. 8b). Identical results were obtained whenautolysis was triggered by other methods (ex-posure to hypertonic sucrose or to freezing andthawing [7]) or in experiments in which theautolytic activities of Triton-ethylenediamine-tetraacetic acid extracts (7) (made from tolerantand lysis-prone cells) were compared.

DISCUSSIONThe mechanisms by which inhibition of peni-

cillin-sensitive bacterial enzymes (or PBP)brings about interference with the growth of abacterial cell is not clearly understood presently(24). Chemically different beta-lactam antibiot-ics can elicit a variety of different morphologicaleffects and distinct growth inhibitory mecha-nisms in the same bacterium (E. coli) (20, 21).There are also examples of the converse situa-tion since different species of bacteria treatedwith the same beta-lactam antibiotic may showa wide variety of physiological responses, suchas inhibition of growth (with only an extremelyslow loss of viability), rapid loss of viability, andloss of viability accompanied by cellular lysis (9,26). Studies on pneumococci and some othergram-positive bacteria have shown that the

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960 KITANO AND TOMASZ

X -17 76 ; | _

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C-80

FIG. 6. Effect of beta-lactam antibiotics on cell shape of E. coli X1776 and its mutants. (A) Control; (B)cephaloridine; (C) mecillinam; (D) cephalexin. Cultures of the bacteria grown at 32°C were exposed to fourtimes the MICs of the various beta-lactam antibiotics for 3 h. After fixation with 2% glutaraldehyde, phase-contrast micrographs were made of the bacteria, using a Zeiss microscope fitted with a Planachromat 100/1.25 phase-contrast oil immersion objective. Kodak Panatomic-X filn was used for the photomicrography.

beta-lactam antibiotic-induced lysis requires theactivity of bacterial autolysins (2, 6, 26). Pneu-mococci in which the in vivo activity of the N-acetyl muramic acid-L-alanine amidase has beensuppressed (by mutation or physiological manip-ulations) show a unique response to penicillintreatment: in such cells the antibiotic causesinhibition of growth (at the normal MIC) andoften the rate of loss of viability is also greatlyreduced, but no lysis occurs (25, 26).We proposed the term "antibiotic tolerance"

for this phenomenon (25), and recent reportssuggest that antibiotic tolerance may not berestricted to laboratory strains of pneumococcibut may also occur among clinical isolates ofStaphylococcus aureus (17) and Streptococcussanguis (10). The involvement of autolytic ac-tivity in the penicillin-induced lysis of E. colihas been repeatedly suggested in the literature,and penicillin treatment of E. coli has beenreported to cause enzymatic cell wall degrada-tion (19) and also an increase in the in situautolytic activity (5). In a recent, more detailedstudy, we found a striking quantitative correla-tion between the efficiency of beta-lactam anti-biotics to trigger autolytic cell wall degradationand the relative affinity of these antibiotics forthe PBP 1 group of E. coli (13). In this com-

munication we describe the properties of threeE. coli isolates that exhibit a tolerant responseto treatment with penicillins and cephalosporins.Each one of the isolates has a defective autolyticsystem.One of the isolates (x2452) exhibits a tolerant

response when grown at 420C and a lytic bacte-ricidal response when grown at 32°C. The othertwo mutants (C-80 and C-254) show tolerance atall temperatures of growth. These E. coli mu-tants resemble the autolysis-defective pneumo-cocci in that each one of them exhibits an ab-normality in their autolytic system: brief treat-ment of the mutants (or of X2452 grown at 4200)with beta-lactam antibiotics does not trigger cellwall degradation, although the same treatmentcauses rapid and extensive wall hydrolysis in theparental bacteria and in strain X2452 grown atthe lysis-permissive temperature (320C). Addi-tional tests have revealed a substantially de-creased autolytic activity in the mutant cells (ascompared with the parental bacteria). However,cells of strain X2452 grown at either 32 or 420Cappear to have comparable (normal) levels ofautolysin activity. Thus, tolerance of mutants C-80 and C-254 is accompanied by an apparent netdecrease in autolysin activity, whereas in X2452only the triggering of autolytic activity seems

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BETA-LACTAM-TOLERANT MUTANTS OF E. COLI 961

X-2452

32 C

42 C

1 2 4 8

Antibiotic concentration (MIC units)FIG. 7. Triggering of autolytic activity by cepha-

loridine. After growth in medium supplemented with[3H]DAP for several generations, cells weregrown inradioisotope-free medium for an additional cell gen-eration. After this period, 1.5-ml portions of the cul-ture were treated with increasing concentrations ofcephaloridine for 20 min (strain x2452) or for 10 min(strain xl776 and its mutant derivatives). Thegrowthtemperature was 320C for all strains except for a

portion of X2452, which was also grown at the lysis-protective temperature of 42°C. For the assay of cell

wall degradation, cells of all strains (including the420C-grown bacteria) were incubated at 32°C afterwashing and resuspension in buffer (1.5 ml). Radio-activity released during2 h ofincubation in the bufferwas counted, and the autolytic activity was expressedas the rate relative to the spontaneous rate ofreleaseof radioactivity from the untreated bacteria. Thislatter value has amounted to about 8% of the totalincorporated radioactivity (13).

thermosensitive. The titration of the total au-tolysin content of E. coli presents considerableproblems since there are several enzyme activi-ties involved (8, 16) and some of these are struc-ture bound. Furthermore, in extracts, some ofthese hydrolytic enzymes may be restricted intheir ability to attack cell walls. For these rea-sons, it was felt that a determination of in situautolysin activity (7) as it was done in our ex-periments may be an appropriate first approxi-mation when comparing autolysin levels of tol-erant (mutant) and lysis-prone (parental) bac-teria.The level of autolysin activity was also deter-

mined in Triton-EDTA extracts (7) preparedfrom the parental cells and from the mutants C-80 and C-254. The mutant extracts showed a

diminished specific hydrolase activity (about 25to 30% that of the extract from parent cells). Inthese assays radioactive DAP-labeled cell walls(murein sacculi [3]) from the parental bacteriawere used as substrate (data not shown). It isnot yet known whether the lowered autolysinactivity in the mutants involves one or more ofthe multiple types of murein hydrolases of E.coli (8, 16).

The mechanism by which interference withcell wall synthesis provokes autolytic cell walldegradation in E. coli is not yet known. In thecase of pneumococci, we proposed that the sui-cidal activity of the autolytic amidase may becaused by a defect that rapidly upsets the invivo control of this enzyme after the addition ofpenicillin to the bacteria (26). This suggestionwas based on the demonstrated release of anautolysin inhibitory agent (Forssman antigen)from pneumococci during penicillin treatment(26). There is no evidence at this time for theexistence of analogous autolysin inhibitors in E.coli. The triggering of in situ autolytic wall deg-radation of E. coli after exposure of the cells tovarious chemical treatments (e.g., cold trichlo-roacetic acid, ethylenediaminetetraacetic acid,high concentration of sucrose [7]) or mechanicaldisruption has been explained as the disruptionof a "barrier" (plasma membrane?) that wassuggested to separate the autolysins of E. colifrom their substrate in the normal cells (7).Rapid release of substantial quantities of lipidsand other membrane components have beendemonstrated during penicillin treatment of sev-eral autolysin-defective gram-positive bacteria(9,11, 12). Preliminary experiments indicate thatpenicillin-induced release of lipid material alsooccurs in some strains of E. coli (A. Tomasz,unpublished data), and it is conceivable thatsuch a process may damage the hypothetical

A B60

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HOURSFIG. 8. Autolytic activity triggered by trichloro-

acetic acid treatment. [3H]DAP-labeled cells weresuspended in 0.01 M Tris-maleate buffer (pH 6.0)containing 10 mM MgSO4 and mixed with an equalvolume of 10% trichloroacetic acid and allowed toreact for 10 min in ice (7). The cells were washedthree times by centrifugation in the buffer, resus-pended in the same buffer, and incubated at 32°C.The radioactivity released into the supernatant so-lution was assayed at different times by theproceduredescribed in Materials and Methods. (A) Cells ofX2452 grown at 32°C (O) or 420C (*). (B) Cells ofX1776 (O) and its mutants C-80 (*) and C-254 (A).Bacteria were grown at 320C.

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962 KITANO AND TOMASZ

barrier separating autolysins and the murein ina manner somewhat analogous to the damagethat may be responsible for triggering by chem-ical treatments. If this were true, then the lackof autolysin triggering in strain X2452 grown at420C may be caused by the presence of a morestable barrier (different membrane composi-tion). Alternatively, cell wall synthesis at thehigher temperature may produce a murein oflow autolysin sensitivity at a critical area of themutant cell wall.

It should be emphasized that the nature ofautolysin triggering is obscure at the presenttime. There is strong evidence implicating thefunctional involvement of both the binding pro-teins 1 (PBP la and lb) (21-23) as well as theautolytic enzyme(s) of E. coli in the penicillin-induced lysis of this bacterium. However, it isnot clear how and why inhibition of PBP leadsto initiation of autolytic cell wall degradation,and the term triggering refers to this poorlyunderstood connection between the functions ofPBP 1 and the control of autolysin activity.Besides the hypothetical barrier function, trig-gering may involve accumulation of cell wallprecursors or even the local modification of themurein substrate, e.g., by introduction of poorlycross-linked material into areas of the cell wallduring penicillin treatment (15).

In the thermosensitive tolerant mutant X2452the heat-sensitive element in the penicillin re-sponse seems to be this hypothetical triggeringprocess since both autolytic activity and thePBP patterns are normal at each temperature(data not shown). The substantially lowered au-tolytic activity and antibiotic tolerance of mu-tants C-80 and C-254 are reminiscent of theproperties of autolysis-defective pneumococci.On the other hand, lysis of the E. coli mutantscan still be induced by treatment with inhibitorsof early steps in cell wall biosynthesis, whereasthe autolysis defective pneumococci are resist-ant to the lytic effect of all cell wall inhibitors(25). It is conceivable that the tolerant pheno-type has a more complex biochemical and ge-netic basis in E. coli than in pneumococci be-cause of the more complex nature of autolysinregulation and the differences in the mode ofcell wall assembly in E. coli and in pneumococci.The morphological changes induced in the

tolerant E. coli during treatment with differentbeta-lactam antibiotics require comment. Itseems that inhibitors ofPBP 2 (mecillinam) andPBP 3 (cephalexin) can elicit their typical mor-phological effects in these cells (i.e., ovoid cellformation and filament formation), whereas theresponse of the tolerant E. coli to inhibitors ofPBP 1 is changed: instead of the appearance of

J. BACTERIOL.

"rabbit ear" forms and lysis, such cells wouldelongate (Fig. 5). These findings support thenotion that in E. coli triggering of autolysinactivity by beta-lactam antibiotics is a specificconsequence of the inhibition of PBP 1. Thetolerant cells appear to be defective in the se-quence of events leading from PBP 1 to autoly-sin triggering; they respond to inhibitors of PBP1 by reversible inhibition of growth (instead ofloss of viability and lysis). These observationssuggest that triggered autolysin activity may bethe cause of viability loss (as well as cell lysis) inE. coli.

It is hoped that these mutants will be helpfulin the analysis of the mechanism of the antimi-crobial effect of beta-lactam antibiotics. A mu-tant of E. coli strain K-12 that is tolerant to lysisby cephalexin has been isolated independentlyby T. Nikaido, S. Tomioka, and M. Matsuhashi(personal communication). No defect in a pep-tidoglycan-lytic enzyme activity has so far beendemonstrated.

ACKNOMWLEDGMENTSThese investigations have been supported by grants from

the National Science Foundation (PCM 7812770) and theNational Institutes of Health (AI 12932). We thank RoyCurtiss III and Dennis Pereira (University of Alabama, Bir-mingham, Ala.) for providing us with strains X1776 and X2452

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