JOURNAL OF BACTERIOLOGY, August, 1966Copyright ( 1966 American Society for Microbiology

Vol. 92, No. 2Printed in U.S.A.

Delayed Lactose Fermentation by EnterobacteriaceaelR. E. GOODMAN2 AND M. J. PICKETT

Department of Bacteriology, University of California, Los Angeles, California

Received for publication 23 December 1965

ABSTRACTGOODMAN, R. E. (University of California, Los Angeles), AND M. J. PICKETT.

Delayed lactose fermentation by Enterobacteriaceae. J. Bacteriol. 92:318-327.1966.-When 171 Citrobacter freundii strains and 14 Paracolobactrum arizonaestrains examined, 51 of the C. freundii strains and 13 of the P. arizonae strains werefound to be delayed or negative lactose fermenters. Of the slow fermenters, 65%yielded rapidly fermenting mutants in cultures undergoing delayed fermentation.Lactose fermentation could generally be hastened by increasing lactose concentra-tions. Many organisms which fermented lactose slowly grew readily on a mediumcontaining lactose as the sole carbon source. Regardless of their ability to fermentlactose, all strains of C. freundii and P. arizonae investigated could be shown to pos-sess 3-galactosidase. Delayed fermenters failed to take up lactose from the culturemedium, whereas prompt fermenters did so readily. The ,B-galactosidases of 12strains of enteric bacteria were studied in crude cell extracts with respect to specificactivity, stability, and activity at varying substrate (o-nitrophenyl-3-D-galacto-pyranoside) concentrations, at varying pH, and in the presence of sodium, potas-sium, and magnesium. The widely varying specific activities and the approximatesimilarity of the Michaelis constants (about 2 X 10-4 M) suggested that the strainsinvestigated produced differing amounts of 3-galactosidase. Moreover, qualitativedifferences in the enzymes provided evidence that these strains synthesized differentmolecular forms of 3-galactosidase. The results suggested that organisms whichferment lactose only after a prolonged delay do so because they possess multipledefects in their lactose-metabolizing machinery.

Fermentation of lactose has long been used todifferentiate between enteric bacteria. Thoseorganisms which ferment the sugar only after adelay ranging from 3 days to many weeks areeasily confused with those which do not fermentat all. Early studies of delayed lactose fermenta-tion (15, 25) led to the hypothesis that the delaywas a result of a nonfermenting culture givingrise to fermenting mutants. Deere, Dulaney, andMichelson (10) found that Escherichia colimutabile, grown in the absence of lactose, pro-duced only small amounts of lactase (,3-galactosid-ase), whereas in the presence of the sugar largeamounts of the enzyme were produced. Even thenonfermenting E. coli type, when grown in thepresence of lactose, possessed considerable

1 Taken from a dissertation submitted by R. E.Goodman in partial fulfillment of the requirements forthe Ph.D. degree, University of California, Los An-geles.

2Present address: Department of Microbiology,School of Medicine, University of Washington,Seattle.

lactase (9). The enzyme was only "active," how-ever, when the cells were disrupted. Thus, theinability of this strain to ferment lactose was duenot to the absence of the lactose-splitting enzymebut to a lack of permeability of the cells to thesugar. This phenomenon is termed "crypticity"(6). Deere's (9) study suggested that the changeto lactose positivity was probably a mutation topermeability. Similar crypticity and mutativefermentation in a paracolon bacillus have beenreported (21, 22). Mutative fermentation oflactose has also been noted with Shigella sonnei(4, 8) and various members of the Salmonellagroup (16).The rapidity of lactose fermentation may be

profoundly influenced by the composition of themedium. Lowe and Evans (22) noted more rapidfermentation of 3 to 5% lactose in media contain-ing 0.1% peptone than in media containing 1.0%peptone. Schafler (31) confirmed this observa-tion, and showed that the phenotypic expressionof lactose-positive mutants may be retarded inpeptone water.

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LACTOSE FERMENTATION BY ENTEROBACTERIACEAE

The concept of the lactose (lac) operon and itsproducts (13, 14) leads to several hypotheses forlate fermentation of lactose. These may beplaced in three groups encompassing permease,,B-galactosidase, and regulatory functions. Thepermease defect might be (i) a molecular form ofpermease which has a low affinity for lactose, (ii)too few "molecules" of permease, or (iii) absenceof permease (y-), in which case the sugar pre-sumably would enter the cells by passive diffusion.These three hypotheses have in common thepredictions that uptake of lactose would beretarded and that in fermenting cultures acidic endproducts would slowly accumulate. Permeaselessstrains might ferment lactose when autolysis ofthe culture occurs, allowing the sugar to come incontact with 3-galactosidase, or fermentationmight be a result of a y- y+ mutation.

Hypotheses pertaining to f3-galactosidase are:(i) among different strains of bacteria, differentmolecular forms of ,B-galactosidase may exist,some of which have a low affinity for lactose(some forms might have a tertiary structurewhich would cause them to be quite labile);(ii) some strains might produce quantitativelyless f-galactosidase than others; (iii) delayedlactose fermentation might occur as a result of az- + z+ mutation. Mutations involving regu-latory functions can be proposed which mightaccount for late lactose fermentation: (i) isi+ or is i- (35); (ii) o00 o+; and (iii) mutationssuppressing o0 (2, 32). Catabolite repression (24,26) too could conceivably be involved, as fermen-tation tests are usually performed in the presenceof peptone.Many of the above hypotheses are not mutually

exclusive; it is likely that in diverse strains dif-ferent defects exist, and even a single strain mightpossess more than one defect. The experimentalwork reported here was intended to assess someof the factors influencing delayed fermentation oflactose by Enterobacteriaceae and to test some ofthe hypotheses mentioned above.

MATERIALS AND METHODS

Organisms. The bacteria studied included 171strains of Citrobacter freundii, 14 of Paracolobactrumarizonae, and one each of E. coli K-12, E. coli ML-3,Salmonella paratyphi B, Shigella sonnei, and a lactose-fermenting strain of Proteus rettgeri. All strains wererapid glucose fermenters.

Media, reagents, and techniques. The 2% lactosemedium was the tableted substrate described by Hoytand Pickett (12). EMB-lac was a mineral-eosin-methylene blue-agar medium containing only lactoseas available carbon. Four fluid media, each containing0.001% bromocresol purple, were designed to test thefermentation of lactose; these were: A, 1% lactose and0.1% peptone; B, 5% lactose and 0.1% peptone; C,

1% lactose and 1% peptone; and D, 5% lactose and1% peptone.

Lactose was assayed by a modification of the an-throne test described by Dische (11), and protein wasassayed by a modification of the procedure of Lowryet al. (23). Crude extracts were obtained from washedcells which had been grown in aerated synthetic me-dium (see Table 1) or Heart Infusion Broth (Difco)supplemented with 1% lactose. The cells were disin-tegrated in a Sagers (30) press, and 30 ml or less of0.02 M sodium phosphate (pH 7.5) was added. Crudeextracts were separated from cell debris by centrifuga-tion at 17,000 X g and were stored at -10 C untilused. Quantitative assay for ,B-galactosidase was per-formed by a modification of Lederberg's (18) proce-dure. The reaction mixture contained 0.00333 Mo-nitrophenyl-,3-D-galactopyranoside (ONPG; Calbio-chem), the required enzyme dilution, 0.05 M sodiumphosphate buffer (pH 7.5), and 0.0016 M MgSO4. Insome experiments, 0.01 M mercaptoethanol was pres-ent. To determine the thermal stability of j-galactosi-dase, samples were diluted with Tris buffer [0.05 Mtris(hydroxymethyl)aminomethane-maleate, pH 7.5]supplemented with sufficient bovine albumin (Armourfraction V) so that each diluted sample contained 4.33mg of protein per ml. The buffer-albumin mixture waspreheated to the inactivating temperature (prior to ad-dition of enzyme). At 3-min intervals, samples wereremoved, diluted into cold buffer, and assayed for,-galactosidase activity. To determine the thermal sta-bility of 8-galactosidase in vivo, bacteria were grownat 25 C in an aerated broth medium (IM-4) consistingof: (NH4)2SO4, 3.0 g; MgSO4-7H20, 0.2 g; KH2PO4,6.12 g; K2HPO4, 9.6 g; CaCl2, 0.01 g; FeSO4c7H20,0.0005 g; disodium succinate, 3.0 g; yeast extract(Difco), 0.3 g; and deionized water, 1 liter. For thefinal 4 hr of growth, 8 X 10-4 M isopropyl4--D-thio-galactopyranoside (IPTG; Calbiochem), as inducer,was added to the culture. The bacteria were harvestedfrom this broth culture by centrifugation and weresuspended in preheated Tris-maleate buffer containing30 jug of chloramphenicol per ml (to prevent enzymesynthesis during determination of thermal stability).Samples were taken at 3-min intervals, diluted intocold buffer, and assayed for ,B-galactosidase activity.Medium IM-4, again containing IPTG, was also usedfor studying the effect of toluene on ,B-galactosidase.

Lactose fermentation. Each inoculum for six differ-ent media was 0.1 ml of a suspension obtained byharvesting the overnight growth on a Kligler IronAgar (KIA) slant with 1 ml of sterile water. The fourbroth media, A to D (4 ml per 13 by 100mm test tube),were observed for 20 days before being discarded asnegative; with all tubes incubated beyond 3 days, theirsteel caps (Morton) were replaced by rubber stoppers.The EMB-lac plates were examined after 3 days ofincubation, and the number of colonies was estimated.The 2% lactose medium was examined at 6, 24, and48 hr. When a broth medium showed delayed fermen-tation, a subculture was plated on EMB-lac or Mac-Conkey agar, and a colony from this was transferredto KIA and, after growth on this medium, was checkedin 2% lactose medium for promptness of (i.e., evi-

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GOODMAN AND PICKETT

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FIG. 1. Fermentative action offive strains of entericbacteria on 1% lactose, 0.1% peptone. Symbols: 0 =

pH ofmedium; A = fi-galactosidase activity. (A) Citro-bacterfreundii strain 17; (B) C. freundii strain 144; (C)C. freundii strain 10; (D) C. freundii strain 142; (E)Paracolobactrum arizonae strain 1.

dence of mutative) fermentation. All incubation was at35 C.

Patterns offermentation. A 400-ml amount of me-dium A (without pH indicator) in a stoppered 500-mlflask was inoculated with 1 ml of a Heart InfusionBroth culture. The flask was incubated at 35 C for 20days, samples being removed at intervals by syringeand needle. Each sample was checked for pH andassayed for both extracellular and cellular ,-galac-tosidase.

Lactose uptake. Washed bacteria, harvested fromHeart Infusion Broth (Difco) supplemented with0.05% NaCl and 0.1 %7 glucose, were suspended in 120ml of IM-4 (above) and divided into four 30-ml por-tions. These were aerated at 25 C for 4 hr, two of themwith 8 X 10-4 M IPTG. The four cultures were cen-trifuged, resuspended in a small volume of IM-4, andadjusted to 8 X 1010 cells per milliliter in a total vol-ume of 1 ml. Chloramphenicol was then added to allfour. The duplicate induced suspensions and one non-induced suspension then received 1 mg/ml of lactose;the fourth served as conti ol. All suspensions were thenincubated at 25 C and assayed at 30 and 60 min forextracellular lactose.

RESULTS

Lactose fermentation. Of 171 strains of C.freundii, 120 fermented 2% lactose promptly, 32were "delayed" (more than 6 hr in the 2% lactosemedium), and 19 were negative after 2 days ofincubation (the tests were routinely discarded atthis time). One strain of P. arrizonae fermentedlactose promptly and 13 were lactose negative.Only three strains failed to acidify any of the

four broth media (A, B, C, and D) within 20days. With 128 pairs of tubes, where the onlydifference between members of a pair was thelactose concentration, fermentation occurredfaster in 5% lactose with 113 pairs (88%7), andwas absent entirely in 1% lactose with 12%cl of thepairs. In no case was acid produced with 1% butnot 5% lactose. Moreover, 58 %O of pairedcultures, in which the only difference betweenmembers of a pair was the peptone concentration,fermented faster in 0.1 %L7 peptone. For 65% of thestrains, presumptive mutants which fermentedlactose faster than the parent strain were isolatedfrom tubes showing acid production.

Patterns offermentation. Twenty-one strains ofbacteria were selected for investigation on thebasis of their differing fermentative reactions inthe above experiment. The criterion for fermenta-tion in the present experiment was attainment of apH of 5.2 in the culture medium. In all cases,there was an initial marked drop in pH observed5 hr after inoculation. This drop was probablynot indicative of lactose fermentation, becauseby 12 hr the pH usually began to rise and by 1day reached a level at which it remained, iffermentation was not taking place, or from which

320 J. BACTERIOL.

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VOL. 92, 1966 LACTOSE FERMENTATION

it again descended, if fermentation occurred.Although in many cases 3-galactosidase waseasily detectable, even in nonfermenting cultures,in no case was a significant amount of enzymedetected in the supernatant fluid. The followingC. freundii strains did not ferment lactose in 20days: 17, 22, 64, 80, 153, 163, 144, 149, and 157.The first six of these strains, of which 17 is anexample (Fig. 1A), also failed to yield detectablefl-galactosidase. The other three, however, ofwhich 144 is an example (Fig. iB), did show f-galactosidase activity which gradually disap-peared during the 20-day period of the experi-ment.The following strains fermented lactose within

20 days and possessed readily demonstrable (3-galactosidase: C. freundii strains 10, 42, 68, 127,18, 57, 89, 107, and 142, and P. arizonae strains1, 3, and 10. The first four, of which C. freundii10 is an example (Fig. 1C), showed a gradual butsteady drop in pH without an appreciable lag,and a concomitant rise in ,B-galactosidaseactivity. However, enzyme activity did notcontinue to rise as the pH dropped, but began todecrease between the 4th and 7th days (Fig. IC).With the remaining eight strains, of which C.freundli 142 (Fig. 1D) and P. arizonae 1 (Fig. IE)are examples, the pH began to drop only after alag ranging from 2 to 12 days (depending on thestrain). The finding of a lag preceding the drop inpH offers support for the hypothesis of autolyticfermentation. Moreover, in all cases j-galactosid-ase activity was detectable before fermentationstarted but rose sharply when thepH began to fall.

Detection of f3-galactosidase. The slowestfermenters and several apparent nonfermenters,collectively referred to as "weak" strains (28),were selected for further study (Table 1). C.freundii strain 2 and P. arizonae strain 15 wereincluded as positive controls, and S. paratyphiB as a negative control. The reactions of theseveral strains on 2% lactose are shown for com-parison. Two of the media contained a high con-centration of lactose (5%) in case any of thestrains possessed an inducible ,B-galactosidase buthad a permeability barrier. All strains of C.freundii and P. arizonae investigated could beshown, by one test or another, to possess a j3-galactosidase (those strains not considered hereproduced, in preliminary experiments, easilydetectable (-galactosidase under a variety ofconditions). Moreover, medium G, similar toKIA but containing nopH indicator, was superiorto the other media for demonstration of theenzyme. Disruption of weak strains with tolueneeither had no effect or an inhibitory one.

Lactose uptake. Evidence of permeabilitydefects was sought by examination of 29 strains

E3Y ENTEROBACTERIACEAE 321

TABLE 1. Detection of fI-galactosidase in Citro-bacter freundii and Paracolobactrum arizonae

grown on various media

Strain

C. freundii 2......P. arizonae 15....Salmonella para-

typhi B ........C. freundii 17.....C. freundii 19.....C. freundii 22.....C. freundii 33.....C. freundii 64.....C. freundii 80.....C. freundii 82.....C. freundii 153....C. freundii 160....C. freundii 161....C. freundii 162....C. freundii 163....C. freundii 164....P. arizonae 14....

Time (hr) ofappearanceof acid in2% lactose

66

24

Time (hr) of appearanceof positive test (yellow)

Medium Medium MediumEb Fb Gb

1 1 0.21

4

124

244

4

324

82424

2448484848968

aA blank space indicates that the organismwas not tested under the given conditions; aminus sign indicates that no ONP was detected.bMedium E: (NH4)2SO4, 6.0 g; MgSO4.7H20,

0.2 g; glycerol, 5.0 ml; lactose, 50.0 g; 0.1 M sodiumphosphate buffer (pH 7.3), 500 ml; and deionizedwater, 500 ml; filter-sterilized. Medium F: Cas-amino Acids (Difco), 20.0 g; yeast extract (Difco),1.0 g; glycerol, 5.0 ml; lactose, 50.0 g; and dis-tilled water, 1 liter; pH 7. Medium G: TrypticaseSoy Broth (BBL) supplemented with 2% agar,1% lactose, and 0.1% glucose.

from several groups of Enterobacteriaceae withrespect to their ability to take up lactose in 30and 60 min from the culture medium. Typicalresults are presented in Table 2. E. coli strains K-12 and ML-3 (cryptic) were tested as positive andnegative permease controls, respectively. It isapparent that K-12, when induced, took up overtwo thirds of the lactose in 1 hr. The noninducedK-12 suspension did not take up the sugar nordid ML-3, whether induced or noninduced. Ofthe strains investigated, only C. freundii 2, P.arizonae 15, Proteus rettgeri 3, and E. coli K-12showed evidence of lactose permeability. All theseare prompt fermenters. The remaining strains(including 17 not shown in Table 2) are slow ornegative fermenters and showed no evidence oflactose uptake. Interestingly, the permease ofProteus was constitutive.

Comparison of fi-galactosidase in crude extractsfrom representative Enterobacteriaceae. Twelve

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TABLE 2. Lactose uptake by various Enterobacteriaceaea

Strain

Escherichia coli K-12................E. coli ML-3 .......................Citrobacter freundii 2...............C. freundii 10.......................C. freundii 13.......................C. freundii 22.......................C. freundii 33.......................Paracolobactrum arizonae 1..........P. arizonae 15......................Shigella sonnei......................Proteus rettgeri 3...................

Induced

30 min

7.621.52.320.219.316.820.518.316.622.47.7

60 min

5.921.82.221.220.822.422.019.413.220.79.2

30 min

8.222.32.4

20.217.319.322.318.815.722.98.5

60 min

5.721.01.8

20.519.620.522.019.713.421.59.0

Noninduced

30 min

20.520.218.319.819.022.521.319.521.622.96.2

60 min

20.620.018.620.719.721.921.122.221.821.06.9

aData are expressed as micrograms of anthrone-positive material (as lactose) in 20 ,uliters of cellsuspension supernatant fluid. If no sugar was taken up by the cells, 20,gg of lactose should be present.

bDuplicate experiments.

TABLE 3. Il-Galact:osidase ictivity ofcrude extracts in the presence and absence of0.01 M mercapeolthanol

Specific activityb Michaelis constant(units/mg of protein) (moles/liter)

ExtractaMercaptoethanol Mercaptoethanol Mercaptoethanol Mercaptoethanol

present absent present absent

Escherichia coli K-12..................... 8,000 4,920 1.4 X 10-4 1.4 X 1O-4Citrobacter freundii 2 224 (57)c 1.6 X 10-4C. jreundii 2 HI ......................... 131 132 1.9 X 10-4 1.4 X 10-4C. freundii 10 HI ..22 2.9 X 10-4C. freundii 10 HI-2....................... 224 141 3.1 X 10-4C. freundii 92 ..20 2.2 X 104C. freundii 92 HI......................... 33 29 1.4 X 10-4C. freundii 107 HI........................ 46 42 3.4 X 10-4 3.2 X 10-4C. freundii C-127 ..225 3.2 X 10-4C. freundii 127 HI........................ 35 29 1.9 X 10-4 1.5 X 10-4Paracolobactrum arizonae 3 HI ..........27 20 2.4 X 10-4 2.1 X 10-4P. arizonae 15 ........................... 287 500 (244)c 2.2 X 104 1.7 X 10-4Proteus rettgeri 3 HI..................... 1,930 1,940 1 .9 X 10-4 1.6 X 10-4

a HI following strain number indicates that the bacteria were brown in Heart Infusion Broth with1% lactose. Otherwise they were grown in medium E of Table 1.

b The unit is that amount of enzyme which hydrolyzes 1 m,umole of ONPG in 1 min under the con-ditions of assay described in Materials and Methods.

c Values in parentheses represent specific activities after 4 months of storage at -10 C.

strains, differing in their ability to (i) fermentlactose, (ii) discharge rapidly fermenting mutants,and (iii) hydrolyze ONPG, were studied. E. coliK-12 was included as a reference organism, andthe Proteus strain was included because it was ofinterest to compare its ,B-galactosidase with thoseof the other enteric bacilli. Results obtainedwith 13 extracts from nine strains are given inTable 3 (three Citrobacter strains, 13, 17, and 22,showed no 3-galactosidase activity in cell-free

extracts regardless of the medium used forgrowth and induction). With most of the enzymesinvestigated here, activity was increased in thepresence of mercaptoethanol. However, C.freundii 2 HI and P. rettgeri 3 HI showed nosignificant difference. (HI after a strain numberindicates that the bacteria were grown in HeartInfusion Broth with 1 % lactose; otherwise theywere grown in medium E of Table 1.) Two of theenzymes lost activity during 4 months of storage

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TABLE 4. Optimal pH values of ,3-galactosidases

S-Galactosidase prepne Magnesium Mapesiumpresent absent

Escherichia coli K-12..... 6.6-6.9 6.9-7.2Citrobacter freundii2 6.6-6.9 6.9C. freundii10 HI......... 6.9 7.1-7.2C. freundii 92............ 6.6 6.9-7.2C. freundii 107 HI.6.0-6.5 6.9-7.2C. freundii 127........... 6.3 6.9 and 7.8Paracolobactrum arizonae

3 HI .................. 6.6-6.9 6.9-7.2P. arizonae 15 ........... 6.9 7.1 and 7.8Proteus rettgeri 3 HI.. 6.6-6.8 6.9

a HI following strain number indicates that thebacteria were grown in Heart Infusion Broth with1% lactose. Otherwise they were grown in mediumE of Table 1.

at freezer temperature (-10 C). C. freundii 2lost about 75% of its activity and P. arizonae 15lost about 50%. All of the other /-galactosidasesappeared to be quite stable. The specific activitiesvaried widely-over two orders of magnitude.Certain strains (e.g., C. freundii 10 and 127)yielded extracts differing 10-fold in activity; noexplanation for this was sought.A convenient comparison for enzymes of like

function is provided by their Michaelis constants(Kin) for a given substrate. Toward this end, theseenzymes were assayed with substrate concentra-tions ranging from 4 X 10- I to 8 X 10-4 M, andapparent Km values for ONPG were calculatedby the method of Lineweaver and Burk (20).Table 3 lists the apparent Km values found. Theywere substantially the same, averaging around2 X 10-4 M. The differences observed in twoextracts of C. freundii 127 were as great as anyobserved between different fl-galactosidases.Thus, the Km values in Table 3 cannot be con-sidered significantly different.The effect of varying pH on enzyme activity

was investigated with the pH of 0.05 M sodiumphosphate buffer ranging from 5 to 8.5. The pHoptima in the presence and absence of 0.0016 Mmagnesium ion are presented in Table 4. In-variably, the presence of magnesium in the reac-tion mixture yielded a lower pH optimum thanthat found when this ion was absent. Thisconfirms a similar observation made with ,B-galactosidase from E. coli ML 308 (28). In thepresent study, when magnesium was omitted allthe ,B-galactosidases had about the same pHoptimum, approximately 6.9 to 7.2. However,enzymes from C. freundii 127 and P. arizonae 15seemed to have a second optimum, 7.8. [Cohn andMonod (7) observed two pH optima, 7 and 7.6 to7.8, for fl-galactosidase from E. coli ML assayed

TABLE 5. Relative activities of f3-galactosidases inthree buffers

Sodium Potas-phos- Potas- slum

P-Galactosidase prepna phe sphois- photwith phos- withneimag ht mag-nesium ~nesium

Escherichia coli K-12. 100 34 64Citrobacter freundii 2..... 100 64 77C. freundii 10 HI......... 100 58 66C. freundii 92 HI......... 100 77 82C. freundii 107 HI........ 100 68 91C. freundii 127.100 73 104Paracolobactrum arizonae3 HI................... 100 52 56

P. arizonae 15............ 100 23 46Proteus rettgeri 3 HI ..... 100 30 60

a HI following strain number indicates that thebacteria were grown in Heart Infusion Brothwith 1% lactose. Otherwise they were grown inmedium E of Table 1.

in potassium phosphate buffer, values which aresimilar to that for C. freundii 127 and P. arizonae15. These authors had no explanation for this butdid not think that it was due to two molecularforms of fl-galactosidase in the preparation.] Inthe presence of magnesium, all enzymes exceptthose of C. freundii 107 HI and 127 had pHoptima ranging from 6.6 to 6.9. The two excep-tions were somewhat lower.

Lederberg (18) noted that sodium ion was abetter activator of ,B-galactosidase than potassiumion for hydrolysis of ONPG in cell-free extractsof E. coh K-12. Enzyme activities of nine extractswere therefore compared in three buffers, all atpH 7.5: (i) 0.05 M sodium phosphate with 0.0016M MgSO4; (ii) 0.05 M potassium phosphate; and(iii) 0.05 M potassium phosphate with 0.0016 MMgSO4. The results, as percentage of maximalactivity, are presented in Table 5. With one orpossibly two exceptions, C. freundii 127 and 107HI, sodium proved to be superior to potassiumas an activator. Moreover, in the potassiumbuffer, magnesium invariably stimulated 3-galactosidase, although the degree of stimulationwas quite variable.Another convenient means of comparing

enzymes is to determine their sensitivity toinactivation by heat. Toward this end, 11 f-galactosidases were investigated with respect totheir rates of inactivation at several temperatures(Table 6). The enzymes differed markedly withrespect to thermostability, ranging from almostcomplete stability at 55 C (E. coli K-12) to 98%inactivation in 30 min at 46 C (C. freundii 10HI-2).

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TABLE 6. Per cent inactivation of,-galactosidasesafter 30-min exposure to several temperatures

i9-Galactosidase Temp of inactivation

prepna55 C 52 C 49 C 46 C 43 C

Escherichia coli K-12... 17 0Paracolobactrum ari-zonae 3 HI ........... 83 21 12

Citrobacter freundii 2HI 98......9 8. ....9 47 9

Proteus rettgeri 3 HI. 100 4C. freundii 127 HI...... 100 53 3C. freundii 127 ....... 100 95 35 0P. arizonae 15.......... 100 99 80 20C. freundii 92 HI 100 91 10C. freundii 10 HI 100 84 40C. freundii 107 HI 100 98 15C. freundii 10 HI-210....._ 98 35

a HI following strain number indicates thatthe bacteria were grown in Heart Infusion Brothwith 1% lactose. Otherwise they were grown inmedium E of Table 1.

Thermal inactivation of f3-galactosidases in vivo.In a preliminary experiment, we found that C.freundii 22 and 82 yielded greater ,3-galac-tosidase activity when induced at 25 C rather than37 C. However, because a cell extract of strain22 possessing 3-galactosidase activity could notbe prepared (regardless of the temperature ofinduction), in vivo experiments were necessary.No attempt was made to prepare active cellextracts of strain 82. Both enzymes were found tobe quite thermolabile. The (3-galactosidase ofstrain 82 was 35% inactivated at 43 C in 3 min,and that of strain 22 was over 90% inactivatedunder the same conditions. At 37 C, the fi-galactosidase of strain 82 was relatively stable,but that of strain 22 was over 90% inactivated in30 min. None of the #-galactosidases studied invitro was as sensitive to heat as were those ofthese two strains. To check the possibility that thein vivo procedure caused a more rapid inactiva-tion of the enzyme than did the in vitro procedure,we tested the f-galactosidases of C. freundii 10and 127 by the former. The enzyme of strain 10proved to be completely stable at 43 and 46 C,although it was quite sensitive to these tempera-tures in vitro. Likewise, the 3-galactosidase ofstrain 127 was quite stable at 43 and 52 C. Thus,although the data from the in vivo experimentsare not directly comparable with those fromexperiments performed in vitro, it appears thatthe former procedure enhances the apparentthermal stability. It follows that the enzymes ofC. freundii 22 and 82 are extremely heat-sensitive,

TABLE 7. 8-galactosidase activity offour strains ofCitrobacter freundii exposed to toluene

for 4 hra

Before After toluene assayStrain toluene

assay 1 min 30 min 1 hr 4 hr

13 41.1 80.0 100 1.6 0.2418 10.5 97.2 100 98.5 97.222 48.3 100 55.1 37.1 1.9182 95.7 100 91.4 100 78.2

a Data are expressed as percentage of maximalactivity.

the former so much so that it would be quicklyinactivated at normal incubation temperatures.

Effect of toluene on /3-galactosidase. We notedabove that toluene inhibited ,B-galactosidaseactivity in several slowly-fermenting strains ofCitrobacter. It was not clear, however, whetherthis effect was due to inhibition of induction or tosensitivity of the ,B-galactosidases to the solvent.Therefore, four strains were induced with IPTG,treated with toluene, and assayed at intervals upto 4 hr. The results are presented in Table 7. Thefour strains showed an immediate increase inenzyme activity when treated with toluene,although with C. freundii 82 this effect wasslight. When toluene was allowed to act forseveral hours, the #-galactosidases of C. freundiistrains 13 and 22 were inactivated and those ofstrains 18 and 82 were resistant. Therefore, thepreviously observed inhibition of ,B-galactosidaseactivity of strain 82 (Table 1) was probably due toinhibition by toluene of enzyme synthesis.

DIscussIoN

Early investigations of delayed lactose fer-mentation led to the idea that the delay was aresult of a nonfermenting culture giving rise tofermenting mutants. At the same time, it wasevident that permeability of the cells to lactosewas of importance. In essence, the experimentsreported here confirm the early work and, inaddition, offer some support for several hy-potheses presented in the introduction.

Permeability defects. Many of the resultspertain to hypotheses involving permeability. (i)Fermentation was often hastened by increasingthe lactose concentration from 1 to 5%. (ii)Fermentation was inhibited by peptone. Thiswould be expected if, as a result of a permeasedefect, lactose was very slowly metabolized andacidic end products were only gradually produced.The acid might easily be neutralized by thepeptone or its catabolites. Additional evidence

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for slow production of acid was obtained whenfour strains showed a gradual drop in pH duringfermentation. (iii) Many strains which did notferment lactose, or did so exceedingly slowly,grew on EMB-lac, a medium providing lactoseas the sole carbon source. Presumably, enoughlactose entered the cells to allow growth but notenough for the accumulation of acidic endproducts. (iv) Many slowly fermenting (or non-fermenting) strains produced easily demonstrablef-galactosidase. (v) The delayed fermentersstudied with respect to lactose uptake, many ofwhich produced easily demonstrable 3-galactosid-ase, were, without exception, permease-negative.The conclusion is inescapable that permeabilitydefects play an important role in retarding lactosefermentation.The autolysis hypothesis was supported by the

finding that eight strains began to ferment lactoseonly after a lag ranging from 2 to 12 days. Withthree of these strains, it was unlikely that muta-tion was involved, because they failed to yieldrapidly fermenting mutants in a previous experi-ment. If autolysis did occur, the ,B-galactosidasewas not released into the medium, but apparentlyan alteration in permeability of the cells tookplace which allowed the substrate to enter andcome in contact with the enzyme. Autolysis hasbeen implicated by Pappagianis and Phaff (27)in delayed fermentation of sucrose by yeasts.They found that sucrase activity remained withthe cell debris and was not released into thesupernatant fluid. A strain of Shigella dysenteriaewas observed by Li, Barksdale, and Garmise (19)to ferment lactose as a result of autolysis mediatedby a carried bacteriophage (T7). A similarphenomenon was observed by Coetzee andHawtrey (5) with sucrose fermentation by astrain of Proteus mirabilis.

fi-Galactosidase. Although f-galactosidase hasbeen investigated in great detail, most reportshave been concerned with the enzymes of E. colistrains K-12 and ML or their derivatives. Depend-ing on the conditions of assay, the Km for ONPGin the presence of at least 0.02 M Na+ is between10-4 and 2 x 101 M (17, 18, 33). Values for theoptimal pH have been reported ranging from 6.6to 7.5, again depending on the conditions of assay(34). Several 3-galactosidases from enteric bac-teria have been described which differ from the E.coli enzyme with respect to one or more charac-teristics (1, 3, 19, 29, 36; Sarkar, Ph.D. Thesis,Massachusetts Institute of Technology, Cam-bridge).The widely varying specific activities and the

similarity of the Michaelis constants reportedhere offer presumptive evidence that the strains ofEnterobacteriaceae investigated produce differing

amounts of ,B-galactosidase. Unfortunately,however, the variation in specific activities of dif-ferent extracts of single strains was too great tomake this any more than an assumption. Yet, it isreasonable to assume that P. arizonae 3 HI, whichgave rise to extract with a specific activity of 27,produced fewer molecules of the enzyme per cellthan did E. coli K-12, which yielded extract with aspecific activity of 8,000. The extracts from strainswhich fermented lactose promptly tended to havehigher specific activities than those from slowfermenters. This supports the hypothesis thatrapidity of fermentation is dependent on theamount of f-galactosidase produced. The rangeof Km observed for the enzymes (1.4 X 10-4 to3.4 X 10-4 M) is consistent with values reportedby other workers for 3-galactosidases of Entero-bacteriaceae (1, 17, 18, 33, 36).

Other findings reveal differences among theenzymes, and we suggest, therefore, that the f-galactosidases considered here represent differentmolecular forms of the enzyme. Whether or notthese differences have relevance for delayedlactose fermentation is debatable. Certainly,strains harboring enzymes as unstable as thoseof C. freundii 13, 17, 22, and 82 might havedifficulty in hydrolyzing lactose, but little can besaid about the differences among the remainingenzymes. We may note parenthetically here thatthe finding of the genetic potential for synthesis of,B-galactosidase in all strains of C. freundii andP. arizonae examined strongly supports use of the"ONPG test" in clinical bacteriology (28).

Catabolite repression as a cause of delayedlactose fermentation is not supported by the workreported here. Although fermentation did occurfaster if the lactose concentration was increasedfrom 1 to 5%, this was probably a result of theovercoming of a permeability barrier rather thana competition between lactose and a repressor.Ability to detect /-galactosidase in slow fer-menters was related to the complexity of themedium employed for induction; this is contraryto what one would expect if these strains werehypersensitive to catabolite repression.Mutation appears from our results to be the

most frequent cause of delayed fermentation. Ofthe various kinds of mutation, the most likely, inview of the results mentioned above regardingpermease defects, is y- y+. However, othermutations proposed in the introduction are alsopossible. Perhaps the most important fact here isthat mutation is not the sole cause of delayedfermentation; from approximately 35% of thelate-fermenting strains investigated, rapid mutantswere not isolated.

Evolution of lactose-negative organisms-

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GOODMAN ANDPICKE1TJ

TABLE 8. Summary of characteristics ofrepresenitative strainis

,6-GalactosidaseCitro- Latoe n-bacter fermen- E " Per-freundiisc measeTounstrain tation O Stability Tolenei

$.. ~~~~~~~~tivity

2 Prompt + Intermediate -

10 Delayed + - Thermolabile17 Delayed - - Poor22 Delayed + - Thermolabile +

accumulation of multiple defects. Why are orga-nisms with defects in their lactose-metabolizingmachinery isolated with such frequency? The lacoperon only has selective advantage when lactoseis present, and, in many adult humans and otheranimals, long periods might elapse when thesugar is not present in the gut. Indeed, the func-tion of the lac operon is to turn off synthesis ofthe lactose-metabolizing enzymes under suchconditions. A lesion in a gene for one of the lacproteins would have no negative selection pres-sure in the absence of lactose. If the sugar wasabsent for very long, multiple lesions mightaccumulate, rendering an organism pheno-typically lactose-negative. Kristensen's (16)investigation of late fermentation of lo% lactoseby various serotypes of Salmonella has relevancefor this theory. He observed acid after prolongedcultivation, ranging from 44 to 217 days, andisolated rapidly fermenting mutants from theslowly fermenting cultures. If the salmonellae,rather than having deletions of the lac genes,merely have multiple genetic lesions, theirfermentation might be explained as a manifesta-tion of multiple reverse mutations (or possiblysuppressor mutations) after prolonged incuba-tion.

It is not unlikely that many of the organismsmentioned in the present report have suchmultiple lesions (defects). Interestingly, a numberof the mutants isolated from slowly fermentingcultures attacked lactose more rapidly than theparent culture, yet still did not produce acid in2% lactose within 6 hr. They could be one-stepmutations which restored single functions instrainF with several genetic lesions.Table 8 summarizes some relevant character-

istics of a few representative strains. An example ofthose strains which probably have a single defectis C. freundii 10. The defect is apparently one ofpermeability (y-) and is presumably corrected bymutation (y- + y+) to yield rapidly fermentingprogeny. C. freundii 17 is cited as an example ofan organism having multiple defects. One of the

lesions is almost certainly one of permeability(y-). The other is open to question. It is not z-in an absolute sense (deletion), because ,B-galac-tosidase could be detected. Possibly the f-galac-tosidase of this strain is "weaker" than theothers studied here, or it may be that C. freundii17 produces quantitatively less enzyme. Anotherpossibility is that the second lesion is il or o°.This would explain the inability of this strainto be induced and yet allow for low levels of 3-galactosidase to be synthesized. C. freundii 22 isanother example of an organism with multipledefects, and in this case their nature is clear. Oneof the defects must be y-; the other is a very un-stable tertiary structure of 13-galactosidase. Thedefects are apparently repairable by mutation.

ACKNOWLEDGMENTSWe are grateful to R. J. Martinez for incisive criti-

cism and advice at all stages of the work, and to LeilaGreene and Nancy Lundh for their capable technicalassistance.One of us (R. E. G.) was the recipient of Public

Health Service predoctoral fellowships GF-14,734,5 Fl GM-14,734-02, and 5 Fl GM-14,734-03 from theDivision of General Medical Sciences. This investiga-tion was also supported by Public Health Servicegrants AI-05208 and AI-05208-02 from the NationalInstitute of Allergy and Infectious Diseases, and byThe Cancer Research Coordinating Committee, Uni-versity of California (grant 501).

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