JOURNAL OF BACTERIOLOGY, Feb. 1971, p. 609-619Copyright t 1971 American Society for Microbiology

Vol. 105, No. 2Printed in U.S.A.

Temperature-Sensitive Mutants of Escherichia coliAffecting A-Galactoside Transport'

MARJORIE CRANDALL AND ARTHUR L. KOCH

Microbiology Department, Indiana University, Bloomington, Indiana 47401

Received for publication 27 October 1970

Six different temperature-sensitive (ts) mutants have been isolated which haveparental ,B-galactoside permease levels at low temperatures but have decreasedpermease levels when grown at high temperatures. These mutants were derivedfrom Escherichia coli ML308 (lacI-Y+Z+A+). After N-methyl-N'-nitro-N'-nitro-soguanidine mutagenesis, ampicillin was used to select for cells unable to grow on

low lactose concentrations at 42 C. Temperature-sensitive mutants were assayed forgalactoside permease activity after growth in casein hydrolysate medium at 25 or 42C by measuring both radioactive methylthio-fl-D-galactoside uptake and in vivo o-

nitrophenyl-ft-D-galactoside hydrolysis. The six conditional isolates have decreasedlevels of galactoside permease which are correlated with decreased growth rates atelevated temperatures. The low permease levels are not due to a temperature labilelacY gene product but rather to a temperature labile synthesis rate of functionalpermease. Some of the mutants exhibit a ts increase in permeability as shown bythe increased leakage of intracellular #-galactosidase and by the increased rate of invivo o-nitrophenyl-fl-D-galactoside hydrolysis via the nonpermease mediated entrymechanism. Preliminary evidence indicates that transport in general is decreased inthese mutants, yet there is some specificity in the mutational lesion since glucosidetransport is unaffected. All these observations suggest that these mutants have tsalterations in membrane synthesis which results in pleiotropic effects on variousmembrane functions.

The galactoside permease is part of a systemwhich catalyzes the transport and accumulationof lactose and other f,-galactosides across thepermeability barrier of the cell (23). This per-mease is a protein (molecular weight, 30,000);reference (12), is coded for by the lacY gene ofthe lac operon (6), and is found bound to themembrane (7). The galactoside permease (alsoreferred to as M protein for membrane protein)acts as a binding protein for galactosides whensolubilized.

Since the galactoside permease is membrane-bound, it would be expected that a change inpermease activity would result if the membranestructure were altered. Membrane structure orcomposition can be altered either genetically orphysiologically. In this paper we describe geneti-cally altered Escherichia coil strains which ap-pear to have a temperature-sensitive (ts) altera-tion in membrane synthesis that affects severalmembrane functions including permease activity.Other workers have described effects on per-

' Presented in part at the 70th Annual Meeting of the American So-ciety for Microbiology, Boston, Mass., 26 April-I May 1970.

mease activity as a result of altering the composi-tion of the membrane. This was done by growingunsaturated fatty acid auxotrophs of E. coli inmedia supplemented with various fatty acids. Anall or none effect on permease activity was foundby Fox (5) who studied induction of the galacto-side permease in an unsaturated fatty acid auxo-troph isolated by Silbert and Vagelos (27). Foxfound that an unsaturated fatty acid (e.g., oleicacid) must be present during induction for func-tional permease to be synthesized. Under theconditions of his experiments the synthesis of theother gene products of the lac operon, ,B-galacto-sidase and transacetylase, proceeds in the absenceof the required growth factor. Since the synthesisof these soluble cytoplasmic proteins proceedsfrom a polycistronic messenger (see reference 3for a recent review) and since the lacY permeasegene maps between these two enzyme markers, itwas concluded that the galactoside permease pro-tein was also synthesized but was not functionalbecause its incorporation into the membrane de-pends on new membrane synthesis which couldnot take place in the absence of the required fattyacid. Fox takes this as evidence that there are no

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preformed and available binding sites on themembrane for the permease protein. His conclu-sion agrees with that of Boniface and Koch (2)who also presented evidence that growth andhence new membrane synthesis is a necessary stepfor the appearance of functional permease afterinduction. This conclusion confirms the first re-port on the galactoside permease by Rickenberget al (23) who showed that the appearance of newpermease after addition of inducer is directly pro-portional to the amount of new growth.Whereas Fox has shown that lipids play a role

in the synthesis of functional permease activity,other workers have shown that the chemicalstructure of the particular fatty acid given assupplement not only affects the physical proper-ties of the membrane but also affects permeaseactivity to varying degrees (25, 28).

Permease activity involves the cooperative ac-tion of several membrane components. It has thusbeen possible to isolate different types of mutantsaffecting each of these components. A ts galacto-side permease mutant has been isolated whose Mprotein is temperature labile (6). This mutant rap-idly loses in vivo permease activity after transferto high temperature, and the isolated M protein,itself, is more thermally labile in vitro than theparental M protein. Another mutant has been de-scribed which is unable to couple metabolic en-ergy to galactoside transport (30). This mutantcan transport galactosides but cannot accumulatethem. The mutants reported here have permeasewith the same temperature stability of the parentin vivo but lose permease activity by dilutionduring growth at elevated temperatures. In thesemutants the synthesis of functional permease is tsand is probably due to a ts alteration in a stepneeded for membrane synthesis. This alterationresults in a variety of ts pleiotropic effects ongrowth, permeability, and transport.

MATERIALS AND METHODSBacterial strains. The parental strain was E. coliML308 (1acI- Y+ZA+). It was chosen because it isconstitutive, shows no sign of reverting to wild-type(lacI+) and has been used for many physiological studiesof transport and energy coupling.

Chemicals. Ortho-nitrophenyl-,B-D-thio-galactopyran-oside (TONPG) was obtained from Cyclo ChemicalCorp. Ortho-nitrophenyl-jl-D-galactopyranoside(ONPG) was obtained from Mann Fine Chemicals,Inc., New York, N.Y. N-methyl-N'-nitro-N'-nitroso-guanidine was purchased from Aldrich Chemical Co.,Inc. Milwaukee, Wis. Sodium ampicillin (polycillin-N)was purchased from Bristol Laboratories, Inc., Syra-cuse, N.Y., a division of Bristol-Myers Co. The radio-active chemicals used were: 14C-methylthio-fl-D-galactopyranoside (TMG) from Schwarz BioResearchInc., Orangebury, N.Y., a-methyl-D-glucopyranoside,

glucose-U-14C (aMG) from Calbiochem, Los Angeles,Calif.; and uracil-2-14C from New England NuclearCorp., Boston, Mass.Media and growth conditions. Minimal M-9 salts

medium, pH 7.0, contained per liter of deionized dis-tilled water: NH4C1, I g; KH2PO4, 3 g; Na2HPO4, 6 g;NaCI, 5 g; and MgSO4-7H2O, 100 mg. Carbon sourceswere autoclaved separately at 10 or 100 times concen-trations and then added to the M-9 salts. Unless statedotherwise, all experiments were done with culturesgrown in M-9 salts medium plus 0.5% casein hydroly-sate [acid-hydrolyzed, salt-free, vitamin-free, withouttryptophan (Nutritional Biochemicals Corp., Cleveland,Ohio)] as the sole carbon source. This medium allowedfor vigorous growth without causing high degrees ofcatabolite repression and, in addition, provided theamino acids required for growth at high temperaturesof some of the mutants. Cultures were inoculated intoliquid medium from stock slants (prepared at 22 to 26C) and grown at 25 C in aerated tubes in a water bathuntil mid-exponential phase (OD at 420 nm = 0.2 to0.8). At this time the culture was either used for an ex-periment, diluted 1/10 or 1/100 to maintain exponen-tial growth, or refrigerated for later use as a liquid in-oculum. Great care was taken to ensure that cells hadbeen growing exponentially for many generations be-cause permease levels are lowest when cells are firstinoculated into fresh medium or when stationary phaseis reached (Fig. 1). Minimal plates contained 1.5% no.2 lonagar (Oxoid) which gave no visible growth unlessa carbon source was added. Cryptic cells (permease-less) were detected on 0.1% succinate minimal platescontaining 10-3 M TONPG which is toxic to 1acY+cells (J. Sadler, personal communication and reference21). The yellow powder was dissolved in the sterilizedmelted agar before pouring the plates. Lactose fermen-tation was assayed on EMB lactose (Levine EMBAgar; Difco Laboratories, Inc., Detroit, Mich.) andLZ (lactose-tetrazolium) plates (26). Lactose fermen-tation at 25 and 42 C of the mutants was scored bycomparing color development to that of the lac+(ML308) and lac- (ML35) control colonies on identi-cal plates.

Permease assays. The galactoside permease wasmeasured by in vivo ONPG hydrolysis and by radioac-tive TMG uptake by methods used by Koch (17). Therate of in vivo hydrolysis of ONPG by intracellular ,B-galactosidase is limited by the rate of ONPG transportby the fl-galactoside permease. The rate of appearanceof yellow product, o-nitrophenol (ONP), was monitoredcontinuously at 420 nm in a Cary model 16 spectropho-tometer with a thermostatted cuvette chamber, an auto-matic sample changer, and a Houston "Omnigraphic"XT chart recorder which had an interface that per-mitted zero suppression and an increased range of sen-sitivity. A blank of the casein hydrolysate medium wasset to zero OD at 420 nm to correct for the yellow colorof the medium. Permease and turbidity measurementswere done on the cell suspension in the culture medium.It was not necessary to wash the cells since after manygenerations of growth in casein hydrolysate medium,the same value for permease activity is obtainedwhether cells are assayed in the spent medium or resus-pended in M-9 salts medium after being harvested.

610 J. BACTERIOL.

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However, there is an inhibitor of the permease presentin fresh casein hydrolysate medium which will causeabout a 25% decrease in activity (data not shown). Thisinhibition does not affect any of the permease measure-ments in this paper except for perhaps the initial twomeasurements in the two experiments shown in Fig. 5-8. Samples of a growing culture were added to tubescontaining chloramphenicol (50 ,ug/ml final concentra-tion) at 28 C. For the permease assays 2.5 ml of thebacterial suspension at 28 C was added to a cuvette alsoat 28 C. After measuring the absorbance at 420 nm ofthe cell suspension, 200 uliters of 25 mM ONPG wasadded, and the suspension was mixed by blowing airthrough the pipette. After an increase of about 0.1 ab-sorbance units at 420 nm, 25 Mliters of formaldehyde(Fisher Scientific Co., Pittsburgh, Pa., 36.6% solutiondiluted I/ 10 and neutralized to pH 7) was added to in-hibit the permease. The rate of formation of ONP wasagain measured. The rate of hydrolysis by the formalde-hyde control was subtracted from the initial measure-ment to give a hydrolysis rate corrected for leaked en-zyme and penetration of ONPG into intact cells via the"cryptic pathway". The rate of change in absorbanceat 420 nm was multiplied by 0.502 to yield Amoles ofONPG hydrolyzed per minute by I ml of culture at 28C. This factor contains the molar extinction coefficientfor ONP, at pH 7.0, plus volume dilution factors. Aunit of permease activity is defined as the amount thathydrolyzes I ,mole of ONPG per minute under theseconditions. The relative permease levels in the mutantsare expressed as the percentage of the parental ML308specific activity control at the same growth stage. Themutant data are normalized in this way rather than pre-senting the specific activities directly because of thefluctuations in permease levels as a function of thegrowth stage (Fig. 1). It was assumed that similarchanges in permease activity were also taking place inthe mutant when grown at the restrictive temperatures.Therefore, a correction was made for this increase inpermease with growth so that the ts effect which pro-duces a decrease in permease with growth could be iso-lated. The actual correction was done in the followingway. For each experiment, an M L308 control was grownunder identical conditions as for the mutants. It wassampled and assayed for permease activity and dryweight concentration. When the mutants reached thissame dry weight concentration, they were then assayed.Usually the control ML308 permease levels deviatedvery little from the actual values given in Fig. I so thatin the few instances that mutant cultures were assayedat a dry weight concentration different from the con-trol, the levels from Fig. I could be used.

Radioactive TMG uptake was measured on the sameculture assayed for in vivo hydrolysis. After bubblingair through the culture for several minutes at 15 C, a0.5-ml sample of culture was added to 0.5 ml of me-dium containing "4C-labeled TMG also at 15 C. Thefinal concentration of TMG during uptake was 5 x l0-8M. After 30 to 60 sec the mixture was diluted with 2ml of M-9 cooled to 0 C and then rapidly filteredthrough type HA membrane filters (0.45-um pore size;Millipore Corp, Bedford, Mass.) held in a filteringapparatus with a stainless-steel chimney precooled to 0C. The collected cells were rapidly washed three timeswith ice-cold M-9 medium and then the filter was trans-

1200

1000

U1 -X - ----------

600-M3

400

200

o0001 0.002 0.005 0.01 0.02 0.05 0.1 0.2 0.5

WEIGHT CONCENTRATION (mg DRY WT/mIl

FIG. 1. Fluctuations in permease as a function ofgrowth stage. At the start of this experiment an expo-nential culture of ML308 growing at 25 C in caseinhydrolysate medium was diluted 1/100 into fresh me-dium at 42 C (0). When this culture reached stationaryphase, it was again diluted 1/100 into fresh medium at42 C (U). Permease assays were done on samples ofthese cultures without washing.ferred to a vial, dried, and dissolved in Bray's scintilla-tion fluid. A zero-time blank was run at 0 C with allcomponents. The radioactivity in the vials was countedin a model LS233 liquid scintillation counter(Beckman Instruments, Inc., Fullerton, Calif.). Count-ing efficiency for 14C was about 70%. The radioac-tivity of the zero-time blank was subtracted from theincubated suspension to correct for nonspecific ad-sorption of label to cells and filter. The corrected ra-dioactivity per milliliter of each sample was divided bythe mg (dry weight)/ml to yield specific activity. Mu-tant data were expressed as percentage of the ML308control at the same growth stage.

,B-Galactosidase assay. Measurement of the total f,-galactosidase content of the cells was done by lysing thecells. Several drops of a lysing mixture (containing onepart each: toluene, 0.02 M MnSO4, 10% sodium dodecylsulfate, and mercaptoethanol) were added to about 10ml of a bacterial suspension. This cell suspension wasthen incubated 15 min at 37 C until it became viscous.This enzyme preparation was then diluted 50-fold, andthe rate of ONPG hydrolysis was measured as above.

Mutagenesis procedure. Cells of E. coli ML308were harvested in exponential phase and treated ac-cording to the procedure of Adelberg et al. (1) with N-methyl-N'-nitro-N'-nitrosoguanidine at a final concen-tration of 100 Ag/ml, in the dark, without aeration, inthe absence of nutrients, for 15 min at 37 C. The cellswere washed three times, quick-frozen, and stored at-70 C. This mutagenesis procedure yielded a highlevel of mutants (about 10% lac-) with good survival(24%).

Isolation of mutants. This isolation scheme was de-signed to select for cells unable to grow on lactose athigh temperature. (i) Segregation of the mutation: Mu-tagenized cells were first grown at 25 C in 1% lactosebroth to allow for expression of recessive phenotypes.(ii) Ampicillin selection: The cells were starved, washed,and diluted to a concentration of 2 x 10" cells/ml in M-

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9 medium containing 0.0034% lactose. After aerating at42 C for 5.5 hr until the turbidity started to increase, azero-time sample was withdrawn, and ampicillin wasadded to a final concentration of 20 jg/ml (B. Moholt,personal communication; Microbial Genet. Bull., p. 8-9, 1967). The ampicillin powder was dissolved in M-9medium just before use and filter-sterilized. Sphero-plasts were not formed by this ampicillin treatment pre-sumably due to the slow growth rate at the low lactoseconcentration. However, the viable count in the ampi-cillin culture did decrease (from 2.6 to 0.4 x 106cells/ml) with a concomitent increase in the frequencyof lac- cells on LZ agar at 42 C (in this particular cul-ture from 1.6 to 9.5%). Mutant ts227 was isolated fromthe zero-time sample. Mutants ts237, 246, and 249 wereisolated from 80-min ampicillin-treated cells. (iii) Recy-cling in ampicillin: Cells treated for 40 min with ampi-cillin were grown in broth, then in M-9 medium plus0.1% lactose at 25 C and were then starved and treatedwith ampicillin as before. Mutants 182 and 183 wereisolated from the second zero-time sample. Mutantsts186, 188, ts189, 190, ts203, 205, and ts219 were iso-lated from the cells recycled in ampicillin for 160 min.(iv) Counterselection: Cells from zero-time samples, one

cycle, or two cycles of ampicillin selection were spreadon 0.1% lactose minimal plates at dilutions which yieldedabout 100 colonies per plate. Plates were incubated for2 days at 25 C to select against nonconditional mutantsand to allow growth of potential ts lethals. Then theplates were transferred to 42 C and incubated another 2days to identify ts growth. After this incubation proce-dure, wild-type ML308 gave rise to large colonies,whereas the cryptic ML35 gave rise to only very smallcolonies. Out of a total of about 6,000 cells plated, 480small colonies were picked with sterile toothpicks andtransferred to a grid arrangement on 0.1 % glucose mini-mal plates. The glucose plates were incubated for 2 daysat 25 C and then used as master plates. (v) Replicaplating: Mutant patches on the glucose master plateswere replicated to duplicate plates of three differentmedia and incubated and scored as indicated: 0.1% suc-

cinate, incubate for 2 days at either 25 or 42 C, scorefor in vivo ONPG hydrolysis on the plates; 0.1% suc-cinate plus 10-3 M TONPG, incubate for 3 days at ei-ther 25 or 42 C, score for growth; LZ agar, incubate foreither 14 hr at 25 C or 10 hr at 42 C, score for lactosefermentation. (vi) Plate assayfor in vivo ONPG hydrol-ysis: A small drop of sterile 25 mM ONPG was droppedon each patch of cells at room temperature, and thetime for the patch to turn yellow was measured. Con-trol replica patches of ML308 turned dark yellow after3 min, whereas permease-negative ML35 turned onlylight yellow. Thus, permease-positive cultures could bedistinguished from cryptic mutants. However, if theplates were incubated too long, permease-negative cellsappeared positive. (vii) Screening: The six replica platesfrom each master were compared to the ML308 andML35 control replica plates. All mutants which ap-peared ts with respect to any assay were picked with a

sterile toothpick, and a single streak was made on anEMB plate. The streak was incubated I day at 25 Cand scored as lac+ or lac-. Then from this first streakwere made additional streaks for single colony isola-tion, and the plate was further incubated but now at 42C. Colonies (lac+ or'lac-) were again recorded. Isolated

colonies were picked and used to inoculate stock stabswhich were grown at 22 to 26 C. From the 480 mutantpatches, 72 isolates (numbered ML308-182 throughML308-253) appeared to have special properties andwere analyzed further. (viii) Assay of isolates: Isolateswhich were lac+ at 25 C were retested on EMB at 25and 42 C. Nonconditional mutants were set aside, andthe final screening was done with liqdid cultures grownin 0.5% casein hydrolysate at 25 and 42 C and testedfor uptake of radioactive TMG and in vivo ONPG hy-drolysis. When the culture reached mid- or late expo-nential phase, chloramphenicol was added to a finalconcentration of 50 gg/ml. Both permease assays weredone on the same culture and within minutes to avoidany possible changes in permease levels.

RESULTS

Classes of mutants obtained. Many differenttypes of mutants resulted from the isolationscheme described above (Table 1). It was ex-

pected that this isolation scheme would haveyielded both ts lethals and several other classes ofts lac- mutants lacking either ,8-galactosidase orthe galactoside permease or the ability to couplemetabolic energy to galactoside transport, butnone of these classes of mutants were found. In-

stead, mutants were found with phenotypes notpreviously reported. It would be expected thatpermease negative assays would be correlatedwith negative fermentation tests on EMB plates.The six isolates which proved to be ts transportnegative mutants were, however, EMB positive at42 C. Although these mutants were lac+ onEMB, they could be distinguished from ML308because they gave rise to smaller colonies at thehigher temperature and the color developmenttook longer. The other seven isolates were EMBnegative yet permease positive in unusual combi-nations. These latter mutants have not been char-acterized further. Preliminary evidence indicatedthat all six ts transport negative mutants and allseven unusual mutants were unable to grow in thepresence of TONPG at any temperature. Thisgrowth inhibition by TONPG may be becausethese mutants are not completely negative crypticmutants and can therefore accumulate enoughTONPG for it to be toxic as it is for lacY+.

Correlation between growth rates and permeaselevels. All six mutants exhibited decreased growthrates on 0.5% casein hydrolysate at the nonper-missive temperature. This slower growth was cor-related with decreased permease levels understeady state growth conditions (Fig. 2). De-creased permease levels were found using bothassays, indicating that the energy coupling systemwas unaffected by this mutation. This findingthat permease levels varied as a function ofgrowth rate among the mutants suggested thatthere was a common process affecting bothgrowth and permease activity which was altered

612 J. BACTERIOL.

TEMPERATURE-SENSITIVE MUTANTS OF E. COLI

to different degrees in the six different mutants.Since both growth and permease activity dependon proper membrane synthesis, it was reasonedthat the alterations in these mutants could beones which affected the membrane. It is conceiv-able that the alteration in each mutant is differentsince it can be envisioned that many differentprocesses could be altered which would affectmembrane synthesis. In fact, it will be shown thateach of these six mutants has quite differentphenotypes although being related in the way

shown in Fig. 2.ts Nutritional requirements. All of the initial

studies were done on cultures grown in M-9 me-

dium containing 0.5% casein hydrolysate as thesole carbon source. When M-9 minimal mediumwas used with 0.2% succinate as the carbonsource, some of the mutants were unable to growat 42 C without the addition of amino acids. Theamino acid requirement was determined for allsix mutants by testing 18 amino acids mixed to-gether in overlapping pools of four or five aminoacids in 0.2% succinate according to the schemein reference 8. It was found that serine, or cys-teine, or both, will allow growth of mutants ts 189,ts219, and ts237 in succinate medium at 42 C.However, in glycerol medium the ts amino acidrequirement of ts189 is abolished. Mutants ts186and ts203 are not auxotrophs, and ts227 growsvery slowly in all media tested.To determine whether the ts serine or cysteine

requirement in tsl89 and ts219 was related to theother ts effects on growth and transport, proto-trophic revertants were isolated from glucoseplates incubated at 42 C. The revertants wereable to grow on 0.2% succinate in the absence ofan amino acid at 42 C, but they still retainedtheir ts permease phenotype, their ts decreasedgrowth rates, and for ts189 revertants the ts en-zyme leakage characteristic. When prototrophicrevertants of ts189 and ts219 were grown incasein hydrolysate medium at 42 C, again, typi-cally low permease levels were obtained as withthe mutants (K. Fife, personal communication).These results indicate that the original ts mutantshad more than one genetic lesion and that the tsamino acid requirement was independent of the tseffects on membrane function. This conclusionwould be in agreement with the fact that nitroso-guanidine frequently produces multiple mutationsper cell (1).

If the ts amino acid requirement is, in fact,independent of the ts membrane effects then itwould be predicted that other amino acid auxo-

trophs should have unaltered permease levels. Totest this idea, we obtained three pairs of serineauxotrophs and their isogenic parents (E. coli Wand W ser-, KXT and KXT ser-, and B and B

613

TABLE l. Phenotypes of 72 isolates resultingfromselection procedure

EM B- PermeaseLactose assaysa

Class Mutants25C 44C 25C 44C

Nonconditional perme- - - - 44 isolatesase negative

Wild type + + + + 15 isolatesIs Transport negative + + + - is1 86, ts 189,

(small) ts203, ts219,ts227, ts237

Others - - + + 182, 188, 246+ - - + 183, 190+ - + + 205_ + + + 249

To assay the galactoside permease both in vivo ONPG hydrolysis andTMG uptake were measured. In all cases the same relative degree of per-mease activity was obtained for both assays of the mutants when resultswere expressed as percentage of ML308 permease levels (units per gramdry weight) at the same growth stage (see Fig. I and 2 and Table 3 and 4).

ser-) from L. Pizer. These strains were grown incasein hydrolysate medium containing inducer at25 and 42 C for many generations and then as-sayed for in vivo ONPG hydrolysis. In all casesthe serine requirers had as much galactosidepermease as the parental control culture (K. Fife,personal communication). Thus, it can be con-cluded that there is no direct involvement ofserine auxotrophy with permease function in themutants reported in this paper or in other inde-pendently isolated mutants.

I0o

(I)

w

-J.'Li.4

0.

S4

6

41

I IML 308

iMUTANT 186

#0 _ 4 MUTANT 203

_ 1 > MUTANT 219

to MUTANT 237MIUTANT 189 MUTANT 227

I0 40 120 160 200 240 28

DOUBLING TIME (min)FIG. 2. Correlation between permease levels and

growth rates for ts mutants at restrictive temperatures.Cultures of ts mutants and parent ML308 were grownat 44 C in casein hydrolysate medium with repeateddilution for S to 10 generations until steady stategrowth rates and permease levels were attained exceptfor mutant ts227 which only doubled twice during thetime course of this experiment. Permease assays weredone on samples of these cultures without washing.Data are expressed as percentage of ML308 permeaselevels. In this experiment the ML308 permease controlwas 477 units/g (dry weight) for in vivo hydrolysis (U)and 12,700 counts per min per mg (dry weight) forTMG uptake (0).

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Increased permeability at elevated tempera-tures. Some of the mutants exhibit a limited in-crease in the amount of release of intracellular i3-galactosidase after many generations of growthat the nonpermissive temperature. This release iscorrelated with a limited increase in the per-centage accessibility (19) of intracellular ,B-galac-tosidase to exogenous substrate. Stated anotherway, both large and small molecules can pass inand out of the membrane of some of the mutantsmore easily after growth at high temperature.Figure 3 illustrates that mutant ts219 has thesame permeability characteristics as ML308 butthat the other mutants exhibit an amount ofleakage of enzyme which is proportional to theincrease in permeability for ONPG. These results

9

8

7wI-

OC.

5oc9

---

0 2 3 4 5 6 7 8 9 10

ACCESSIBILITY (%)

FIG. 3. Correlation between release of f3-galactosi-dase and increase in membrane permeability at restric-tive temperatures. Same experiment as Fig. 2. Samplesof these cultures were centrifuged, and the cells were

resuspended in fresh casein hydrolysate medium con-

taining 50 ,ug of chloramphenicol/ml. The supernatantfluids of the cultures of Fig. 2 were assayed for released,3-galactosidase. Samples of the suspensions of resus-

pended cells were assayed for in vivo ONPG hydrolysisin the presence offormaldehyde which inhibits the galac-toside permease. Another sample of this suspension was

treated with the toluene mixture to measure the totalintracellular f3-galactosidase. The percentage release of,8-galactosidase during growth at restrictive tempera-tures was calculated by dividing the jI-galactosidasefound in the supernatant fluid by the total intracellular,3-galactosidase. The percentage accessibility (19) ofintracellular enzyme to extracellular substrate was cal-culated by dividing the rate of ONPG hydrolysis informaldehyde-treated cells by the total intracellular 1l-galactosidase. The rate of ONPG hydrolysis by washedformaldehyde-treated cells of ML308 was 32 units/g(dry weight), the total amount of intracellular f3-galac-tosidase was 7,000 units/g (dry weight), and the totalamount of extracellular f3-galactosidase was 2S units/g(dry weight).

indicate that the small amount of release of Il-galactosidase is not due to complete lysis of a fewper cent of the cells; rather, it is a reflection of asmall increase in the permeability of all the cellsin the population. If 5% of the cells lysed, itwould be expected that 5% of the total enzymewould be in the medium but that the remainderof the cells would exhibit a low percentage acces-sibility characteristic of the parental type. This ismore direct evidence that, at least with mutantsts189 and tsl86, the membrane which is synthe-sized during growth at high temperature is al-tered.Permease stability. The decreased permease

levels in the mutants after growth at high temper-atures could be due to either a temperature labilecomponent of the lactose transport system or to atemperature-sensitive synthesis of the permeasesystem. To determine which alternative obtained,the mutants ts 189 and ts219 were grown at per-missive temperature and then transferred to hightemperature. The decay of permease activity (16,23) was followed as a function of time undernongrowing conditions. It was found that per-mease synthesized by the mutants at 25 C was asstable to high temperature as is wild-type per-mease (Fig. 4). lt is important to emphasize thatduring this experiment the membranes of mu-tants grown at 25 C remained as impermeable asdid the membrane of the parent after transfer to44 C. This is shown by the fact that no leakage of,3-galactosidase was obtained under these non-growing conditions at 44 C even with ts 189 whichnormally releases ,B-galactosidase after growth atelevated temperatures. This indicates there is adifference in membrane structure, at least ints189, depending on the temperature of growth.The average half-time of decay of permease ac-tivity at 44 C for the mutants ts189 and ts219under nongrowing conditions was 37 and 40 min,respectively, whereas for ML308 it was 39 min.[These permease half-lives at 44 C are consider-ably less than the half-life reported for ML308 at37 C (300 min) measured under similar condi-tions (16).] Thus, the loss of permease activity inthe mutants during growth at elevated tempera-tures was not due to a temperature-labile compo-nent of the permease system. This conclusion iscompatible with the results presented in the nexttwo sections which show that when cells aretransferred to high temperatures, the loss of per-mease activity is due to a decreased rate of syn-thesis of new functional permease transport sys-tems and a dilution out of preexisting ones withgrowth.

Calculations of the rate of synthesis of per-mease activity. After mutants ts189 and ts219have been grown in casein hydrolysate mediumat 44 C for several generations, the permease

0 MUTANT 1866-

5-

4 MUTANT 1894-

3 MUTANT 219

2 /ML 308 /2 /I/M MUTANT 203

0 9

614 J. BACTERIOL.

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-I 20 MUTANT 219

z r blML308

b MUIANT 18910 6

z04

zw0z00w

a. 2

0 60 120TIME AT 44 C (min)

FIG. 4. Permease decay at restrictive temperaturesunder nongrowing conditions. Cultures of ts mutantsand parent ML308 were grown at 25 C in casein hydro-lysate medium, harvested, resuspended in M-9 mediumwithout a carbon source containing 50 jig ofchloramphenicol/mi, and aerated at 44 C for 2 hr. Thepermease values were 132, 94, and 153 units/g (dryweight)for ML308, tsl89, and ts219, initially.

levels reach steady state values of 10 to 20% ofparental levels (Table 2 and 3). Since the half-time of permease decay is the same in the mu-tants as in the parental type (see previous sec-tion), it was of interest to determine whether thedecreased levels were due to a decreased rate ofsynthesis of new permease or to the longer genera-tion time which might allow more time for thesame amount of permease synthesized to decay.Two equations are necessary to determine the

rate of synthesis of permease. Equation 1 relatesthe change in permease content of the culturewith time to the difference between the rate ofsynthesis of permease and the rate of decay ofpermease.

dP/dt = Sw - kP (1)where P = permease activity (units), t = time(minutes), S= rate of synthesis of permease perunit weight of cells (units/g dry weight), w =weight of bacteria (g dry weight), k = (In 2)/(Ts,a) = permease decay rate constant (1/min)where T112 is the half-life of permease decay(minutes). Equation 2 states that during balancedgrowth the change in permease content with timeis proportional to the growth rate and to theamount of permease activity present at any time.

dP/dt = XP (2)

TABLE 2. Synthesis rate ofpermease at restrictivetemperaturesa

Permease Doubling Dhecaf SynthesisStrain levels time time" rate'

(units/g) (min) (min)

Parent ML308 579 33 39 22.5Mutant ts189 101 75 37 2.85Mutant ts219 93 77 40 2.45

a Steady state growth rates and permease levels atrestrictive temperatures taken from the experiment ofFig. 5.

b Half-time of permease decay at restrictive tempera-tures under nongrowing conditions was calculated byaveraging data obtained from the experiment of Fig. 4.

c Rate of synthesis of permease was calculated byusing equation 3 and the data presented in this table.

TABLE 3. Expression of the lac operon during steadystate growth at restrictive temperaturesa

,8-Galactoside permease levels ,B-Galactosidaselevels

Strain ONPG ONPGhydrolysis TMG uptake hydrolysis

in vivo in vitro

(% of ML308)' (% of ML308)p (units/g)Parent ML308 100 100 7,000Mutants

ts186 91,93,88 78,72,74 7,100ts237 34, 22, 45 21, 19, 22 8,800ts203 35, 24, 34 41, 24, 58 11,000ts219 11, 21, 7 28, 42, 13 5,900ts189 15, 19, 10 12, 14, 9 4,800ts227 14, 16, 12 5, 10, 0 Not done

a Values for permease levels from three different cul-tures are given to show the amount of fluctuation ob-tained. All cultures were grown in casein hydrolysatemedium at 42 to 44 C for many generations.

b Both permease assays are calculated as the per-centage of ML308 permease levels (units per gram, dryweight) at the same growth stage. Typical ML308 con-trol permease levels of cells grown at 44 C ranged from400 to 600 units/g (dry weight).

where X = In 2/ T2 = the growth rate constant(1/min) where T2 is the doubling time of the bac-teria (minutes). By equating 1 and 2 and re-arranging terms, it is seen that

S = (X+ k)P/w (3)When the experimental values were substitutedinto equation 3, it was found that when tsl89 andts219 attain steady state growth and permeaselevels characteristic of restrictive conditions, theysynthesize permease at 13 and 11% the wild-typerate, respectively (Table 2). Thus, the rate of syn-thesis of this transport system is more reduced athigh temperatures than is the rate of synthesis ofother cellular proteins. This ts synthesis of per-mease activity could result from a ts enzyme ac-tivity involved in the synthesis of a membrane

615VOL. 10S, 197 1

CRANDALL AND KOCH

lipid or protein component of the permease trans-port system. However, we must leave open thepossibility that this mutation results in an in-creased lability of the permease protein prior toincorporation into the membrane, at which timeit develops parental stability.

Absence of a regulatory alteration. Repressionof the lactose operon is not involved in thesemutants since it is found that when the low steadylevels of permease activity are reached in themutants at the restrictive temperature, f,-galac-tosidase levels are as high as in the constitutiveparent (Table 3).

Dilution of permease by growth at restrictivetemperatures. When mutants are grown at 25 C,the permease levels are similar to the parentalstrain (Table 4). The deviation from the parentallevel in the various mutants is real and reproduci-ble, but its basis is not clear. When the mutantsare transferred from 25 to 44 C, the decrease inpermease levels at restrictive temperatures can beexplained in terms of decreased rates of synthesisof new permease concomitant with a dilution outof preexisting permease as a function of growth.The situation is shown most clearly with mu-

tant tsI89. This mutant, which becomes leakyafter considerable growth at high temperature,starts losing permease activity immediately aftertransfer to high temperature according to thetheoretical curve based on dilution by new growth(Fig. 5). The theoretical curve was calculated byassuming that immediately after transfer to hightemperature the new, low, steady-state rate ofpermease synthesis was assumed, and then, aftereach cell division, each cell would have half of thepermease which had been synthesized at 25 C.The mutant ts219, which retains parental

membrane permeability characteristics at hightemperatures, shows quite different kinetics sinceit actually shows an initial increase in permeaselevels after transfer and growth at higher temper-

TABLE 4. Permease levels during steady state growth atthe permissive temperaturea

Permease levels

Strain [ONP hydrolysis TMG uptake

in vivo

(% of ML308) (% of ML308)Parent M L308 100 100Mutants

ts186 86, 86, 80 85, 103, 78ts237 131, 159, 103 76, 90, 62ts203 94, 102, 86 109, 123, 94ts219 69, 67, 70 81, 84, 77ts189 72, 72, 70 67, 99, 56ts227 167, 171, 162 123, 135, III

a Footnotes same as for Table 3 except cultures were

grown at 25 C. Typical ML308 control levels at 25 Cranged from 200 to 400 units/g (dry weight).

-J

w5,)

4w

90

sol

70

60

504

40

30

20

10

0 2 3 4 5 6 7NUMBER OF GENERATIONS

FIG. 5. Loss ofpermease as a Junction of growth atrestrictive temperatures. Cultures of ts mutants andparent ML308 were grown at 25 C in casein hydroly-sate medium and then diluted 1/10 into fresh caseinhydrolysate medium at 44 C. After several generationscultures were again diluted 1/10 to maintain exponen-tial growth. ML308 control permease levels increasedfrom 171 to 579 units/g (dry weight) during the course

of this growth experiment.

TABLE 5. Doubling time after a temperature "shift-up"

Preshift Initiala Steady state"Culture 25 C - 44C 44C

(min) (min) (min)Parent ML308 89 34 33

30 28Mutant ts189 108 _ 55 75Mutant ts219 109 _ 44 77

35 60

a Initial doubling time after a temperature shift up.Cells were grown at 25 C and then diluted into freshcasein hydrolysate medium at 44 C.

I Final steady-state doubling time achieved afterabout two generations of growth in casein hydrolysatemedium at 44 C after a temperature shift-up.

atures before losing permease activity. A laterexperiment with ts219 gave the same result, andin both experiments the point at which permease

activity was lost corresponded to midexponentialphase [0.03 mg (dry weight)/ml in casein hydrol-ysate medium] rather than being related to thenunmber of generations of growth. This was alsothe point on the growth curve where the growthrate decreased from its initial high rate to the lowsteady state growth rate at 44 C (Table 5). Whenthese kinetic experiments were repeated in glyc-erol minimal medium (containing serine plus cys-teine) or in casein hydrolysate medium which hadbeen autoclaved for a shorter length of time, bothmutants showed an immediate dilution out ofpermease with growth according to theoreticalpredictions. At this time it is not understood whatdifferences exist between these media. However,since other workers studying permease use cells

-x < MUTANT 219

\\ a

- ...THEORETICAL AND MUTANT 189

_I*I*

616 J. BACTERIOL.

TEMPERATURE-SENSITIVE MUTANTS OF E. COLI

grown in casein hydrolysate, it is pertinent topoint out here how different media can affectpermease levelsAlthough growth rates were measured by tur-

bidity increases and not by colony counts, thedecreased growth rates are probably not due toloss of viability since these cells, when dilutedinto fresh medium at high temperature, show nolag in growth and continue to grow at the slower,steady-state growth rates characteristic of restric-tive conditions. This is in contrast to the situationwith fatty acid auxotrophs. When they use uptheir fatty acid supplement, the turbidity stopsincreasing, the viable count drops, and the cellslyse (10).The data presented in Fig. 5 can be expressed

in another way which is also instructive. The in-crease in permease concentration in a cultureduring growth can be plotted against the increasein dry weight concentration (23). In this plot theslope is the differential rate of net permease syn-thesis. When the data for mutants ts189 and 219were plotted in this way (Fig. 6), it was seen thatthe net differential rates of permease synthesiswere in fact lower than in the parent ML308.This graphic result confirms the previous calcula-tions based on the low, steady-state growth ratesand permease levels after many generations athigh temperatures.

Gain of pernease during growth at permissivetemperatures. Resumption of maximal rates ofpermease synthesis did not occur immediatelyafter transfer of ts219 back to low temperatures

0.100

2

0.0600

0.040_/

ML 30

0.020 _

MUTANT 19

MUTANT 2tn

I-0 0.04 0.06 0.12

WEIGHT CONCENTRATION (mg DRY WT./ml)

0.1X

FIG. 6. Plot of permease synthesis as a function ofgrowth after a temperature shift up. Same experimentas Fig. 5.

IC

-a

0

f) E

*¢_ 70

6e

-J

wJ 4

wH 3.22al

)O

0 1 2 3 4

NUMBER OF GENERATIONSFIG. 7. Gain ofpermease as a function ofgrowth at

permissive temperatures. Cultures of mutant ts219 andparent ML308 were grown at 44 C in casein hydroly-sate medium until steady-state permease levels were

reached, then diluted 1/10 into fresh casein hydrolysatemedium at 25 C. ML308 control permease levels de-creased from 372 to 270 units/g (dry weight) during thecourse of this growth experiment.

0.060

0.040-

0.02 0.04 0.06 0.06 0.10 0.12 0.14 0.WEIGHT CONCENTRATION (mg DRY WT./ml)

FIG. 8. Plot of permease synthesis as a function of

growth after a temperature shift down. Same experi-

ment as Fig. 7.

(Fig. 7 and 8). The theoretical curve was calcu-

lated by assuming that immediately after transfer

to low temperature the steady state rate of per-

mease synthesis characteristic of 25 C was as-

sumed and that at each cell division permease

levels would increase by an increment equal to

half of the difference between the final, steady-

state permease level at 25 C and the permease

level at the previous division. This lag in the re-

sumption of maximal rates of permease synthesis

after a temperature shift down might be ex-

plained if the membrane synthesized at 44 C will

not allow incorporation of new permease mole-

30MUTANT 219 M

50-- - THEORETICAL

1.0so - /AI

WO /50/

0

20C l l5

617VOL. 105, 197 1

I

CRANDALL AND KOCH

cules synthesized at 25 C. Then, a period of newgrowth and hence new membrane synthesis wouldbe required before new functional permease couldbe detected.

Transport of other compounds at high tempera-ture. The mutant isolation scheme was designedto select for cells with decreased ability to trans-port f3-galactosides at high temperature. Whenaccumulation of other classes of compounds wasmeasured by using is 189 and ts219, it was foundthat accumulation of uracil was also ts as com-pared to the parent. In contrast, the uptake ofaMG and glucose is unchanged in these twomutants. These results again indicate that themutational alteration in these mutants affects avariety of processes when cells are grown at hightemperatures. However, there is a measure ofspecificity in this pleiotropic mutation since, inthis case, glucose transport is unaffected.

DISCUSSIONSix different ts mutants have been isolated

which appear to have altered membrane synthesisat high temperature. This results in decreasedgrowth rates, decreased permease synthesis andactivity, decreased generalized transport, and in-creased permeability. In the discussion of these tspleiotropic membrane mutants, it is first neces-sary to eliminate from consideration several al-ternative explanations of the lesion responsiblefor this ts phenotype. These pleiotropic mutantscannot have resulted from a deletion in the lacYgene since the s-galactoside transport system ispresent and functional at 25 C. The mutation hasprobably not affected the lacY gene product sincethe galactoside permease has the same tempera-ture stability as the parent. The mutation has notaffected the expression of the lac operon since ,B-galactosidase is present at the same levels as inthe constitutive parent. The mutation has not af-fected the phosphotransferase system since gluco-side transport is unaffected. Although several ofthese mutants are also ts amino acid auxotrophs,their nutritional requirement was shown to beunrelated to the defect in permease synthesis byprototrophic reversion analysis.The physiological and kinetic studies presented

here strongly indicate the existence of a new genewhich affects lactose utilization indirectly by itseffect on the synthesis of functional permease ac-tivity. The major results which led to this pro-posal are as follows. Although the mutants ex-hibit a ts permease phenotype, they synthesizepermease which decays at the same rate as theparental permease at high temperatures. Thisconclusion is substantiated by the observationthat permease activity is lost under restrictiveconditions only by dilution during growth. Thefindings which implicated a ts alteration in mem-

brane synthesis are the ts decreased permeaselevels, the ts increased permeability characteris-tics of some of the mutants (tsl86 and ts189), thets decreased generalized transport characteristics,and in ts 189 and ts219 altered fatty acid composi-tions (P. Ray, personal communication). Thesechanges in the fatty acid compositions of tsl89and ts219 were unrelated to the expected decreasein unsaturation that occurs after growth at hightemperatures (4, 9, 20) and which were found inthe parent. In the absence of genetic analysis theproposal of a new gene can be only tentative.However, the existence of another locus affectingpermease function would be predicted a priorisince there are two steps involved in permeasesynthesis: the first being synthesis of the poly-peptide coded for by the lac Y gene and thesecond being insertion of this permease proteininto the growing membrane. If there was an al-teration which affected the membrane bindingsite for the permease, then this would by defini-tion affect the synthesis of functional permease.

Although at this point the exact nature of thets membrane alteration reported here is notknown, it is worthwhile to compare it to othermutants which exhibit similar pleiotropic effects.A mutant of E. coli has been reported which isdefective in membrane synthesis and which has a10-fold increased Km for the enzyme L-glycerol-3-phosphate acyltransferase which synthesizesphosphatidic acid (15). It will grow slowly in theabsence of glycerol-phosphate and at near par-ental growth rates when the medium is supple-mented with this membrane constituent. Thismembrane mutant and its parent were obtainedfrom L. Pizer and were grown in either the pres-ence or absence of glycerol-phosphate. No signifi-cant difference in induced permease levels wasobtained although the mutant grown in the ab-sence of its required substrate had slightly lowerpermease levels (K. Fife, personal communica-tion). Another membrane mutant has been de-scribed (J. E. Cronan, T. K. Ray, M. K. Wassef,and P. R. Vagelos, Fed. Proc., p. 342, 1970)which is ts and has a thermolabile L-glycerol-3-phosphate acyltransferase, the same enzyme de-scribed above. This mutant ceases growth andphospholipid biosynthesis immediately upon shiftto nonpermissive temperatures. It would be ofinterest to know whether permease activity orother membrane functions are altered in thismutant. A conditionally defective lipid mutanthas been isolated (C. F. Fox, personal communi-cation) which ceases phospholipid synthesis whenglycerol is removed from the medium. In thismutant there is a pleiotropic defect in the syn-thesis of functional permease but not its function.The last aspect to be discussed and potentially

the most interesting to investigators studying

618 J. BACTERIOL.

TEMPERATURE-SENSITIVE MUTANTS OF E. COLI

other transport systems is the observation thatwhereas generalized transport is decreased ints189 and ts219, glucoside transport is unaffected.This observation adds strength to the argumentthat glucose transport is fundamentally quite dif-ferent from galactoside transport. For example,glucose is phosphorylated during transport (24)whereas j3-galactosides are not (11, 13), and, inaddition, accumulation of galactosides is abol-ished by uncouplers of oxidative phosphorylation,whereas glucose accumulation can be stimulatedby these energy poisons (reviewed in 13, 17, and29). Yet, despite these differences, the two per-

mease systems do, in fact, interact. For example,glucose inhibits galactoside transport (29), andthe reciprocal inhibition can also be demon-strated if the cells are grown in a fast glucosechemostat (A. L. Koch, unpublished observa-tions). Interactions such as these have led to theformulation of models proposing competitionbetween the two different transport systems for a

substance present in limiting amounts variouslycalled "transporter" (14, 17) or "common fac-tor" (29) or perhaps even HPr (18). Thiscommon factor may be the same element referredto as "limiting factor" (5) or phospholipid "car-rier" (22) proposed for the galactoside transportsystem. Since such interactions between transportsystems must take place at the level of the mem-brane and since the membrane and also galacto-side transport has been altered in these mutants,it might be possible by further study of thesemutants to make greater progress in identifyingthe components involved in the membrane pene-

tration step for galactosides.

ACKNOWLEDGMENTS

This investigation was supported by a fellowship (5 F02 A129663) to

M. A. Crandall from the National Institute of Allergy and Infectious Dis-eases and by National Science Foundation grant GB-7022.

LITERATURE CITED

1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Optimal con-

ditions for mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine in

Escherichia coli K12. Biochem. Biophys. Res. Commun. 18:788-

795.2. Boniface, J., and A. L. Koch. 1967. The interaction between per-

meases as a tool to find their relationship on the membrane.

Biochim. Biophys. Acta 135:756-770.3. Carter, T., and A. Newton. 1969. Messenger RNA stability and po-

larity in the lac operon of Escherichia coli. Nature (London) 223:

707-709.4. Esfahani, M., E. M. Barnes, Jr., and S. J. Wakil. 1969. Control of

fatty acid composition in phospholipid of Escherichia coli: response

to fatty acid supplements in a fatty acid auxotroph. Proc. Nat.

Acad. Sci. U.S.A. 64:1057-1064.

5. Fox, C. F. 1969. A lipid requirement for induction of lactose transportin Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 63:850--855.

6. Fox, C. F., J. R. Carter, and E. P. Kennedy. 1967. Genetic control of

the membrane protein component of the lactose transport system of

Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 57:698-705.7. Fox, C. F., and E. P. Kennedy. 1965. Specific labeling and partial pu-

rification of the M protein, a component of the #-galactoside trans-port system of Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 54:891-899.

8. Glover, S. W. 1968. The induction, isolation and analysis of auxo-trophic mutants, p. 17-21. In R. C. Clower and W. Hayes (ed.),Experiments in Microbial Genetics. Blackwell Scientific Publ., Ox-ford.

9. Haest, C. W. M., J. DeGier, and L. L. M. van Deenen. 1969. Changesin the chemical and the barrier properties of the membrane lipids ofE. coli by variation of the temperature of growth. Chem. Phys.Lipids 3:413-417.

10. Henning, U., G. Dennert, K. Rehn, and G. Deppe. 1969. Effects ofoleate starvation in a fatty acid auxotroph of Escherichia co/i K- 12.J. Bacteriol. 98:784-7%.

11. Kashket, E. R., and T. H. Wilson. 1969. Isolation and properties ofmutants of Escherichia coli with increased phosphorylation of thio-methyl-,6-galactoside. Biochim. Biophys. Acta 193:294-307.

12. Kennedy, E. P. 1969. Studies on the lactose transport system in Esch-erichia co/i. J. Gen. Physiol. 54:91s-93s.

13. Kennedy, E. P., and G. A. Scarborough. 1967. Mechanism of hydrol-ysis of o-nitrophenyl-,6-galactoside in Staphylococcus aureus and itssignificance for theories of sugar transport. Proc. Nat. Acad. Sci.U.S.A. 58:225-233.

14. Kepes, A. 1960. etudes cinetiques sur la galactoside-permease d'-Escherichia coli. Biochim. Biophys. Acta 40:70-84.

15. Kito, M., M. Lubin, and L. 1. Pizer. 1969. A mutant of Escherichiaco/i defective in phosphatidic acid synthesis. Biochem. Biophys.Res. Commun. 34-454-458.

16. Koch, A. L. 1963. The inactivation of the transport mechanism for,5-galactosides of Escherichia co/i under various physiological condi-tions. Ann. N.Y. Acad. Sci. 102:602-620.

17. Koch, A. L. 1964. The role of permease in transport. Biochim. Bio-phys. Acta 79:177-200.

18. Kundig,W., F. D. Kundig, B. Anderson, and S. Roseman. 1966. Res-toration of active transport of glycosides in Escherichia coli by acomponent of a phosphotransferase system. J. Biol. Chem. 241:3243-3246.

19. Leive, L. 1968. Studies on the permeability change produced in coli-form bacteria by ethylenediaminetetraacetate.J. Biol. Chem. 243:2373-2380.

20. Marr, A. G., andJ. L. Ingraham. 1962. Effect of temperature on thecomposition of fatty acids in Escherichia coli. J. Bacteriol. 84:1260-1267.

21. MUller-Hill, B., L. Crapo, and W. Gilbert. 1968. Mutants that makemorelac repressor. Proc. Nat. Acad. Sci. U.S.A. 59:1259-1264.

22. Nikaido, H. 1962. Phospholipid as a possible component of carriersystem in,5-galactoside permease of Escherichia co/i. Biochem.Biophys. Res. Commun. 9:486-492.

23. Rickenberg, H.W., G. N. Cohen, G. Buttin, and J. Monod. 1956. Lagalactoside perm6ase d'Escherichia coli. Ann.Inst. Pasteur. (Paris)91:829-857.

24. Roseman,S. 1969. The transport of carbohydrates by a bacterial phos-photransferase system.J. Gen. Physiol. 54:138s-180s.

25. Schairer, H.V., and P. Overath. 1969. Lipids containing trans-unsat-urated fatty acids change the temperature characteristics of thio-methyl galactoside accumulation in Escherichia coli. J. Mol. Biol.44:209-214.

26. Signer, E. R.,J. R. Beckwith, and S. Brenner. 1965. Mapping of sup-pressor loci in Escherichia co/i.J. Mol. Biol.14:153-166.

27. Silbert, D. F., and P. R.Vagelos. 1967. Fatty acid mutant of E. colilacking a 5-hydroxydecanoyl thioester dehydrase. Proc. Nat. Acad.Sci. U.S.A. 58:1579-1586.

28. Wilson, G., S. P. Rose, and C. F. Fox. 1970. The effect of membranelipid unsaturation on glycoside transport. Biochem. Biophys. Res.Commun. 38:617-623.

29. Winkler, H. H., and T. H. Wilson. 1967. Inhibition of,-galactosidetransport by substrates of the glucose transport system in Esche-richia coi. Biochim. Biophys. Acta 135:1030-1051.

30.Wong, P. T. S., E. R. Kashket, and T. H. Wilson. 1970. Energy cou-pling in the lactose transport system of Escherichia coli. Proc. Nat.Acad. Sci. U.S.A. 65:63-69.

619VOL. 105, 1971