MUTATION TO NEOSTIGMINE RESISTANCE IN A CHOLINESTERASE-CONTAINING PSEUDOMONAS'

BARBARA W. SEARLE AND AVRAM GOLDSTEIN

Department of Pharmacology, Stanford University School of Medicine, Palo Alto, California

Received for publication October 26, 1961

ABSTRACT

SEARLE, BARBARA W. (Stanford UniversitySchool of Medicine, Palo Alto, Calif.) ANDAVRAM GOLDSTEIN. Mutation to neostigmineresistance in a cholinesterase-containing Pseudo-monas. J. Bacteriol. 83:789-796. 1962.-In astrain of Pseudomonas fluorescens containing aninducible cholinesterase, the activity of thatenzyme is rate-limiting for growth when acetyl-choline is the sole source of carbon or nitrogen.Under these circumstances, neostigmine, acholinesterase inhibitor, becomes a growth in-hibitor.A neostigmine-resistant mutant was isolated,

and the properties of its cholinesterase werecompared with those of the wild-type enzyme.There were no differences in penetration of cellsby inhibitor, rate of enzyme-inhibitor combina-tion, affinity of inhibitor or substrate for thecholinesterase, or protective effect of substrateupon the enzyme. However, the mutant con-sistently formed cholinesterase at about twicethe wild-type rate. Mutation, in this case, ap-pears to result in a specific change in the differ-ential rate of enzyme biosynthesis. The relation-ship of this change to neostigmine resistance isdiscussed, and it is suggested that the effectobserved here may be prototypic of a generaltype of mechanism responsible for acquired drugresistance.

A strain of Pseudomonas containing an induc-ible cholinesterase was isolated in this labora-tory (Goldstein and Goldstein, 1953; Goldstein,1959). The organism was shown to utilize acetvl-choline (AcCh) for growth by first splitting the

I Material in this paper was part of B. W.Searle's dissertation submitted to Radcliffe Col-lege in partial fulfillment of requirements for thePh.D. degree. A preliminary account of this workhas been presented elsewhere (Searle and Gold-stein, 1957).

ester bond. Its hydrolytic activity was shown tobe sensitive to the well-known cholinesteraseinhibitor, neostigmine (Prostigmin, kindly sup-plied by Hoffmann-LaRoche, Inc., Nutley,N. J.).

Theoretically, one mechanism of acquireddrug resistance in microorganisms could be agenetic change leading to biosynthesis of an en-zyme with altered relative affinities for inhibitorand substrate. Evidence for such a mechanismhas been provided by Davis and Maas (1952)and, more directly, by Saz and Martinez (1956,1958). However, in but few instances has theaction of a growth inhibitor yet been localizedto an effect upon a single well-characterized en-zyme whose properties in wild-type cells anddrug-resistant mutants could be compared.

Since growth of the Pseudomonas could bemade to depend upon its cholinesterase activity,and since this enzyme could be inhibited at will,the organism seemed well suited for exploringmechanisms of drug resistance involving altera-tions in the bacterial cholinesterase. This paperdescribes the conditions under which neostigmineinhibits growth, the isolation of a neostigmine-resistant mutant, and some studies directed to-ward elucidating the mechanism of the acquireddrug resistance. It was found that the resistantmutant forms a cholinesterase indistinguishablefrom the wild type, but does so at a higher rate.Thus, the genetic change conferring neostigmineresistance in this case appears to affect primarilyan aspect of the induction mechanism controllingthe differential rate of cholinesterase biosyn-thesis.

MATERIALS AND METHODS

The organism, a strain of P. fluorescens (ATCC11150), was maintained on mineral agar con-taining AcCh. Growth experiments were donein mineral medium buffered at pH 7.1, in flasks(50 ml in 250-ml flask, 200 ml in 1-liter flask)at 30 C on a reciprocal shaker. The medium had

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the following composition (mmoles/liter): KCl,10; Na2SO41OH20, 1; MgSO4 7H20, 0.4;Na2HPO4, 40; NaH2PO4-H2O, 22. Glucose,when added, was at a final concentration of0.27%; NH4Cl, 0.05%; water-washed agar,1.5%. AcCh was employed as the bromide (re-crystallized); choline chloride and sodium ace-

tate were used as indicated. Neostigmine methyl-sulfate and bromide were used interchangeably.

Cultures used in this study were started withsamples of another culture growing activelyunder the same conditions. Growth was followedturbidimetrically on a Klett-Summerson colorim-eter with a no. 54 filter (more suitable than theusual blue filter because of the presence, undersome conditions, of a yellow-green pigment inthe culture media). Readings were convertedto dry weight by means of a calibration curve.

Especially when (as in most of our experiments)AcCh was the sole source of carbon or nitrogen,growth followed a generally linear course ratherthan an exponential one. This may only reflecta very short exponential phase of growth. Morelikely, it arises from the dependence of growthupon cholinesterase activity (discussed later)and a diminished rate of cholinesterase synthesisduring active growth (Goldstein, 1959). Growthcurves have been represented here on linearrather than logarithmic ordinates.AcCh was measured according to Hestrin

(1949), choline (Ch) by a slight modification ofBeattie's (1936) method, protein N according toLowry et al. (1951).

Cholinesterase activity was measured by C02release in the Warburg respirometer at 30 C, inbicarbonate-buffered salt medium, pH 7.3, in95% N2 and 5% C02. (This medium contained(g/liter): NaCl, 9; KCI, 0.3; MgCl2 6H20, 0.46;NaHCO3, 2.1.)AcCh was added from the side-arm to yield a

concentration of 20 ,moles/ml in the reactionmixture. In some experiments, hydrolysis ofAcCh was determined by serial sampling withthe Hestrin (1949) method, at 37 C, 0.01 M

phosphate buffer, pH 6.0, initial AcCh 3.5,tmoles/ml. The Hestrin assay is more sensitive,and is carried out at optimal conditions foractivity of the bacterial cholinesterase; theWarburg procedure is more suited to followingthe course of hydrolysis, and for inhibitionstudies. Enzyme activity is expressed as ,umolesAcCh hydrolyzed per hr per mg dry wt of cells.All data are corrected for spontaneous hydrolysis.

Under growth conditions, sterile media initiallycontaining 2 ,umoles/ml AcCh lost about 10%of the ester through splontaneous hydrolysis in24 hr.

Intact cells were assayed after harvesting atfull growth, washing, and resuspending toappropriate bacterial density. For extractionand purification of enzyme, washed cell suspen-sions were treated in a 1 0-ke Ravtheon sonicoscillator for 15 min, andl centrifugedl at 20,000 Xg for 25 min. The chlolinesteirase activity wasquantitatively recovered in the supernatant.Further treatment w-ith protamine sulfate(0.06%) after adjustment to 4 mg cell equivalent/ml precipitated material absorbing at 260 mA,as well as 60% of the protein N, but left allenzyme activity in the supernatant. Ammoniumsulfate then precipitated other protein, but noenzyme activity, at 40% saturation; all enzymeactivity finally appeared in the sediment at 75%saturation. Ammonium sulfate was removed bydialysis in the cold, without loss of enzyme activ-ity. Cholinesterase activity in a typical experi-ment was 29 units per mg protein N in the sonicextract supernatant, and 500 units per mg pro-tein N in the final solution.

RESULTS

Growth and its inhibition by neostigmine.Growth on AcCh and on acetate (Ac) pluscholine (Ch), and the effects of supp)lementationwith ammonia and glucose, are shown in Fig. 1.Total growth was the same oni Ac + Ch as onAcCh when these were supplied alone. Addedammonia had no effect but added glucose greatlyincreased the total growth. Growth rate wasalways greater on Ac + Ch than on AcCh, wasunaffected by added ammonia, and was increasedby added glucose. Ac C is not utilized for growthby this organism.

Figure 2 compares the course of growth withdisappearance of substrate from the medium,for growth on AcCh ancd on Ac + Ch. AcCh wasutilized for growth as fast as it disappearedfrom the medium. Ch disappeared much morerapidly than AcCh, and growtth on Ch wasfaster than on AcCh. However, Ch was notutilized for growth as rapidly as it disappearedfrom the medium.The above findings indicate that the cells are

able to assimilate the products of AcOh hydroly-sis faster than they are made available bycholinesterase action, i.e., cholinesterase activity

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90-

'3 60-

30-30

10 20 30 10 20 30 10 20 30

Time in Hours

FIG. 1. Growth of wild-type cells on acetylcholineand on acetate plus choline. Concentrations ofAcCh, Ac, and Ch, 2,moles/ml; NH4Cl, 0.05%;glucose, 0.2%. 0 = AcCh; A = Ac + Ch.

150

100

-._

50

0

10 20 30

Time in Hours

FIG. 2. Growth of wild-type cells and substratedisappearance. Initial substrate concentration:5.5 ,umoles/ml. Solid curves = growth; brokencurves = substrate disappearance from medium.* = Ch; 0 = AcCh.

is rate-limiting for growth when AcCh is thesole substrate and also when AcCh is supple-mented by either ammonia or glucose. On Ac +Ch the growth rate is determined by the rate atwhich Ch C can be utilized (since added am-

monia is without effect); this is faster than therate of AcCh hydrolysis. Ch N is incompletelyutilized unless additional C (as glucose) is fur-nished. In the presence of glucose, Ch N iscompletely utilized and an excess of C remains,since addition of ammonia (not shown) stillfurther increases growth rate and total growth.When cholinesterase activity is rate-limiting

for growth, a cholinesterase inhibitor should act

10 20 30 0 10 20 30

Time in Hours

FIG. 3. Inhibition of growth of wild-type cells byneostigmine. Concentration of AcCh or Ch; 2,umoles/ml. Neostigmine concentrations (M): A = none;0 = 104; A\ = J-3; * = S X 10-3; 0 = 10-2.

as a growth inhibitor, but not otherwise, andthis is indeed the case (Fig. 3). Neostigmine at10-s and 3 X 10-3 M inhibited growth on AcChbut not on Ac + Ch; only at higher concentra-tions was some inhibition obtained on the lattersubstrate. We have also found that the sameneostigmine concentrations inhibit the oxidationof AcCh but not of Ac + Ch by washed wholecells. Moreover, cells grown in the absence of aninducer and containing barely detectable cho-linesterase levels oxidize Ac + Ch but not AcCh.Thus, hydrolytic cleavage is a necessary firststep in the utilization of AcCh by the cells,and the growth-inhibitory effect of neostigmineat concentrations lower than 10-2 M is attribut-able to its inhibition of the bacterial cholinester-ase.The inhibition of growth by neostigmine

could be prevented by increasing concentrationsof AcCh (Table 1). A similar concentration-dependent protection of cholinesterase by sub-strate has been demonstrated in experimentswhere neostigmine inhibits AcCh hydrolysis bywashed whole cells or cell-free extracts (cf.Table 4).

Isolation and properties of the neostigmine-resistant mutant. Cells were plated heavily ontonutrient agar. After 24 hr, the confluent growthon each plate was replicated by means of velvet(Lederberg and Lederberg, 1952) onto a pair ofmineral agar plates containing AcCh (2.5,umoles/ml) and 102 M neostigmine. This in-hibitor concentration in agar completely pre-vented surface growth when AcCh was the sub-strate but not when Ac + Ch was present

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TABLE 1. Protection by acetylcholine against growthinhibition by neostigmine

Linear growth rate*Initial AcCh Per cent of

Control Neostigmine control growth(10-3 ii)

JAmoles/ml

2 3.1 0.9 295 4.4 2.4 558 4.1 3.0 73

16 5.5 5.0 91

* Turbidity units per hr, measured between5 and 15 hr. All cultures were growing at maximalrate during this interval.

instead. Three days later a few colonies hadappeared on each plate. In one pair of replicaplates there were eight colonies on one, nine onthe other, and seven of these were in identicalpositions on both plates. In another pair therewere 11 and 13, with 11 in identical positions.Thus neostigmine-resistant clones were presenton the nutrient agar master plates, havingarisen by spontaneous mutation in the absenceof neostigmine.One of the colonies on a neostigmine plate was

chosen as the source of the resistant mutantwhose properties are compared with those of thewild type in the experiments to be described.The mutant was maintained on AcCh agar andremained stable in the absence of neostigmine.In liquid medium containing AcCh alone, themutant was less inhibited by neostigmine thanwas the wild type (Table 2).We sought to explain the resistance of the

mutant on the basis of poor penetration ofneostigmine into the cells or some qualitativedifference in enzyme-inhibitor-substrate interac-tion within the cells. (That the cholinesterase wasintracellular was shown during its purification.After sonic disruption and centrifugation at20,000 X g, all activity was found in the super-natant.) Neostigmine combines with the bac-terial cholinesterase very slowly. As previouslyshown for mammalian plasma cholinesterase(Goldstein, 1951, 1952), inhibition is muchgreater after preliminary equilibration of in-hibitor with enzyme than when the inhibitor isadded in the presence of substrate. The slow rateof combination of neostigmine with bacterialcholinesterase is shown in Table 3 for wild-typeand mutant cells. The degree of inhibition ul-timately achieved in the absence of substrate is

TABLE 2. Differential effect of neostigmine upongrowth of wild-type and mutant cells in

liquidl medium

Linear growth rate*Neostigmine concn --- --

Linear growth rate*

Wild type Mutant

M

0 3.3 3.510-3 0.8 1.610-2 0.2 0.4

* Turbidity units per hr, measured between5 and 15 hr. All cultures were growing at maximalrate during this inter val. AcCh (2 ,.moles/ml)was the sole source of carbon and nitrogen.

TABLE 3. Kinetics of enzyme-inhibitor combination*

Enzyme activity (umoles mg per hr)

Time of Wild type Mutantaddition of

AcChInhib- Percent Inhib- Per centControl ited of con- Control ited of con-

trol trol

min

45 0.77 0.71 85 0.94 0.68 73105 0.57 0.35 60 0.78 0.44 57150 0.56 0.26 50 0.78 0.42 54210 0.47 0.20 42 0.74 0.29 40

* Intact washed cells. Cholinesterase activitywas measured manometrically. AcCh (5 ,umoles/ml) was tipped in from side-arm after differenttimes of incubation of cells with neostigmine(1.5 X 10-6 M) at 37 C. After 21 hr at 5 C with thisconcentration of inhibitor, the activity was 36%of the control for both cell types.

shown as a function of neostigmine concentra-tion for both strains in Fig. 4. Con(litions of thegrowth experiments were mnore closely simulatedwhen AcCh and neostigmine were added simul-taneously (Fig. 5); here the impressive protectionafforded by substrate is apparent. The concen-tration dependence of tbis p)rotection is shown inTable 4.The data presented above reveal no real dif-

ferences between wild-type and mnutant cellswith respect to the rate of inactivation of theircholinesterases by neostigmine, the affinity oftheir cholinesterases for neostigimine, or theinfluence of AcCh upon the inhibitory efficacyof neostigmine. Since these experiments wereall performed with wvhole-cell suspensions, it isalso evident that the nultant presents no barrier

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a.)-

0

aDCL)

ICO0

A

80 _

60~~~60

40~~~~~~2

20

O0 '_-75 -6.5 -5.5 -4.5

Log of NeostigmineConcentration

FIG. 4. Inhibition of cholinesterase activity. Cellspreincubated with neostigmine for 21 hr at 5 C, fol-lowed by manometric assay of enzyme activity.0 = wild-type cells; A = mutant cells.

a)

0

1-5

c

0

C-)

'4-

0

a)

a1)0-

I(Uu

80_0

60 _ A

40 8

20

a

-3.5 -2.5 - I.5

Log of NeostigmineConcentration

FIG. 5. Inhibition of cholinesterase activity,protection by substrate. AcCh (5,moles/ml) addedsimultaneously with neostigmine in the manometricassay. 0, 0 = wild-type cells; O, A = mutantcells.

to neostigmine penetration. In other experiments,enzyme-inhibitor-substrate affinities and com-

bination kinetics were measured in sonic extractsof wild-type and mutant cells; again no differ-ences were found.The inhibition of bacterial cholinesterase ul-

timately attained with neostigmine is completelyirreversible, as shown by the following. When10-3 M neostigmine was added simultaneouslywith AcCh, the cholinesterase activity was onlyslightly inhibited (Fig. 5). However, if the cells

TABLE 4. Effect of substrate concentration onenzyme inhibiton*

Enzyme activity (Amoles/mg per hr)

AcCh con- Wild type Mutantcentration

Ini-Per cent Inhib- Per centControl itheid- of con- Control ited of con-itd trol trol

,umoles/ml

1 1.63 0.39 24 2.52 0.67 262.5 1.69 0.53 31 2.84 0.83 294 1.42 0.58 41 2.56 0.94 375 1.40 0.58 41 2.33 1.00 43

5 1.22 0.50 41 2.52 0.93 3710 1.03 0.51 50 2.35 1.18 5050 0.87 0.53 61 2.00 1.24 62100 0.82 0.53 65 2.02 1.28 64

* Intact cells grown in Roux bottles. AcCh andneostigmine (10-1 M) were tipped in simultaneouslyfrom side-arm. One experiment with lower andone with higher substrate concentrations areshown.

were exposed to this same inhibitor concentra-tion for only a few minutes without protectionof substrate, inhibition was found to be completewhen AcCh was added. Under these conditions,increasing the AcCh concentration to the limitof solubility could not relieve the inhibition.Similarly, when substrate was added after 90min incubation of cells in 10-6 M neostigmine,even increasing the AcCh concentration ten-fold had no influence on the degree of inhibition.Prolonged dialysis of inhibited cells againstAcCh failed to relieve the inhibition, althoughthe cholinesterase activity of control cellssurvived such dialysis without significant loss.Finallv, dialysis of inhibited against uninhibitedcells failed to reveal any significant dissociationof neostigmine in either the wild or mutantstrains (Table 5).We also attempted to compare affinities of

the partially purified wild-type and mutantcholinesterases for AcCh. It was found, however,that with decreasing AcCh concentration theenzyme activity slowly increased (as substrateinhibition was relieved) down to the limit ofsensitivity of the available methods. In this wayKm could only be estimated as <1 X 10-3 M.Within the range that could be explored, therewas no difference between wild-type and mutant

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enzymes. By using an aromatic analogue ofAcCh (3 - acetoxyphenyl - trimethylammoniumbromide, kindly supplied by Hoffmann-LaRoche,Inc., Nutley, N. J.), we were able to measuresubstrate hydrolysis by ultraviolet spectro-photometry (Goldstein, unpublished observa-tions) down to about 10-5 M. Km for the aromaticsubstrate was found to be 1.0 X 104 M in thecase of both enzymes.

There is one respect in which the two celllines do differ: under most growth conditions thecholinesterase activity of the mutant was nearlydouble that of the wild type (Table 6). This wasexplored more thoroughly in experiments in

TABLE 5. Dialysis of inhibited cells againstuninhibited cells*

Enzyme activity (jmoles/mg per hr)

Dialysis Wild type Mutantconditions

Before After Before Afterdialysis dialysis dialysis dialysis

Control ..... 1.26 1.23 1.81 1.72Cells out-

side bag.. 1.26 1.02 1.81 1.58Cells in bag. 0.05 0.05 0.11 0.14

* Cells were incubated for 1 hr at 37 C with10-4 M neostigmine, then washed. Samples ofinhibited and control uninhibited cells wereassayed for cholinesterase activity by the Hestrinmethod. Inhibited cells (10 mg in 6 ml) were placedinside the bag, uninhibited cells (143 mg in 92ml) were placed outside. The dialysis was per-formed on a magnetic stirrer for 17 hr at 5 C.The control was a sample of uninhibited cellsmaintained under the same conditions in aseparate vessel.

which the enzyme w-as induced in cultures ofhigh bacterial density (2 mg/ml) under conditionsthat did not permit net growth. Here the spec-

trophotometric assay was employed. A typicalresult is shown in Table 7. The basal cholines-terase level of the mutant was nearly twice thatof the wild type. Duriing 24 hr after addition ofCh, the enzyme activity in both cell types in-creased nearly 100-fold. The higher activity ofthe mutant was not only observed in intact cells;approximately twice as much activity was ex-

tractable from mutaint as from wild-type cellsin the procedure that resulted in 17-fold puri-fication with respect to total protein N. Thus,mutant cells synthesize cholinesterase at nearlytwice the wild-type rate under basal conditionsand also in response to inducer. Other experi-ments have shown that the same difference be-tween the two strains occurs at all inducerconcentrations. The change is sl)ecific for cho-linesterase; under all these conditions mutantand wild-type cells did not differ in the rate atwhich they oxidized Ch.

DISCUSSION

In the experiments reported here, the growthof the organism was made to depend upon theactivity of a particular enzyme, an induciblecholinesterase. Since Ae.Ch was the sole source

of carbon and nitrogen, and since the organismwas capable of utilizing Ch faster than it couldhydrolyze AcCh, the cholinesterase activity was

rate-limiting for growth. Under these conditions,as expected, a cholinesterase inhibitor, neostig-mine, became a growth inhibitor. A mutant was

then selected for its ability to grow despite the

TABLE 6. Enzyme activity of wild-type and mutant cells grown under various conditions*

Enzyme activityGrowth substrate Growth conditions

Wild type Mutant

Choline Roux bottle 1.01 ± .06 (10) 2.09 i .04 (10)Shaker 0.80 .06 (4) 1.67 .18 (4)

Acetylcholine Roux bottle 0.48 .03 (6) 1.04 .07 (6)Shaker 0.66 .04 (4) 0.79 .04 (4)

Acetate + choline Roux bottle 0.63 .06 (2) 1.31 .10 (2)Shaker 0.62 .04 (2) 0.92 .04 (2)

* Substrate concentration 10 ;moles/ml. Cells harvested at full growth. Enzyme activity measuredmanometrically. Activities are j,moles AcCh hydrolyzed/hr per mg cell dry wt i standard error. Fig-ures in parentheses indicate the number of determinations.

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TABLE 7. Induction of cholinesterase in wild-typeand mutant cells without net growth*

Turbidity Enzyme activity Activity(Klett units) (jumoles/hr per mg) ratio

(mutant/Wild- wild-

Time type Mutant Wild-type Mutant type)

hr

0 490 490 0.027 0.049 1.81 490 490 0.050 0.091 1.82 490 490 0.110 0.215 2.04 485 485 0.276 0.579 2.16 485 485 0.558 0.945 1.78 0.777 1.26 1.6

24 500 510 2.43 4.07 1.7

* Wild-type and mutant cells were grown inmineral-glucose medium with Casamino acids,harvested, washed thoroughly, and resuspendedin Roux bottles in mineral medium with Ch (10-2M) at a bacterial density corresponding to maximalgrowth under these conditions. Cell samples werewithdrawn periodically, washed, and assayedspectrophotometrically for cholinesterase activitywith an aromatic substrate. (See text.)

presence of an otherwise inhibitory concentrationof neostigmine.

It may be that other mutants can arise whosecholinesterases differ qualitatively with respect torelative affinity for AcCh and neostigmine. Inthe mutant described here, no such differencecould be demonstrated. Neostigmine was shownto have good access to the enzyme within mutantas well as wild-type cells, and the enzyme-inhibitor-substrate interactions were in allrespects the same in both cell types. However,the mutant had consistently higher cholinesteraseactivity per mg than did the wild type.A higher enzyme activity should not by itself

suffice to confer resistance to the growth-inhibi-tory action of neostigmine. We know that agiven concentration of neostigmine will causethe same fractional inhibition of cholinesteraseactivity in wild-type and mutant cells, and thisin turn might be expected to cause the samefractional inhibition of growth in both celltypes. The explanation of resistance in the mu-tant would seem to be that, by increasing itscholinesterase without any change in its Ch-oxi-dase system, it becomes able to hydrolyze AcChmore rapidly than the liberated Ch can beutilized. In the wild type, on the other hand,the cholinesterase is strictly rate-limiting forgrowth on AcCh. In this sense, the mutant may

be said to contain "excess" cholinesterase.Neostigmine is then without effect upon growthuntil a concentration is attained which inhibitsall "excess" enzyme of the mutant cells; thencholinesterase activity becomes rate-limitingfor growth again, and its further inhibition leadsto a lower growth rate.We do not know how general is the mechanism

of drug resistance evidenced here. Similar find-ings have been reported by Broquist et al. (1953)and by Nichol, Zakrzewski, and Welch (1953)with respect to amethopterin-resistant Strepto-coccus faecalis. Assuming that mutation canaffect the differential rate of biosynthesis ofconstitutive as well as inducible enzymes, ageneral mechanism may be imagined wherebygrowing cells can become drug resistant by in-creasing to "excess" their content of the specificenzyme upon which a growth-inhibiting drugacts. Thus in any essential sequence

eW eX

where e1 is inhibited by a drug and W-*X israte-limiting for growth, resistance could be con-ferred by a mutation leading to expansion ofel, so that the rate of a subsequent step (e.g.,X--Y) would become limiting for growth.

ACKNOWLEDGMENT

This work was supported by grants E-1059and CY-2797 from the U. S. Public HealthService.

LITERATURE CITED

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BROQUIST, H. P., A. R. KOHLER, D. J. HUTCHISON,AND J. H. BURCHENAL. 1953. Studies on theenzymatic formation of citrovorum factor byStreptococcus faecalis. J. Biol. Chem. 202:59-66.

DAVIS, B. D., AND W. K. MAAS. 1952. Analysis ofthe biochemical mechanism of drug resistancein certain bacterial mutants. Proc. Natl.Acad. Sci. U. S. 38:775-785.

GOLDSTEIN, A. 1951. Properties and behavior ofpurified human plasma cholinesterase. III.Competitive inhibition by prostigmine andother alkaloids with special reference to differ-ences in kinetic behavior. Arch. Biochem.Biophys. 34:169-188.

GOLDSTEIN, A. 1952. A relationship between the

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rate of attaining equilibrium and the velocityconstant of the reverse reaction in certainenzyme-inhibitor systems. Experientia 8:442-444.

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GOLDSTEIN, D. B., AND A. GOLDSTEIN. 1953. Anadaptive bacterial cholinesterase from a

Pseudomonas species. J. Gen. Microbiol.8:8-17.

HESTRIN, S. 1949. The reaction of acetylcholineand other carboxylic acid derivatives withhydroxylamine, and its analytical applica-tion. J. Biol. Chem. 180:249-261.

LEDERBERG, J., AND E. M. LEDERBERG. 1952.Replica plating and indirect selection of bac-terial mutants. J. Bacteriol. 63:399-406.

LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-

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NICHOL, C. A., S. F. ZAKRZEWSKI, AND A. D.WELCH. 1953. Resistance to folic acid ana-

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SAZ, A. K., AND L. M. MARTINEZ. 1956. Enzymaticbasis of resistance to Aureomycin. I. Differ-ences between flavoprotein nitro reductasesof sensitive and resistant Escherichia coli.J. Biol. Chem. 223:285-292.

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SEARLE, B. W., AND A. GOLDSTEIN. 1957. Neostig-mine resistance in a cholinesterase-containingPseudomonas: a model for the study of ac-

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