JOURNAL OF BACTERIOLOGY, Jan. 1968, p. 52-57Copyright © 1968 American Society for Microbiology

Vol. 95, No. IPrinted in U.S.A.

Galactosidase Activity of Lactose-positive Neisseria'WILLIAM P. CORBETI AND B. WESLEY CATLIN

Department of Microbiology and Immunology, Marquette University School of Medicine;Milwaukee, Wisconsin 53233

Received for publication 23 October 1967

The chromogenic substrate o-nitrophenyl-3-D-galactopyranoside (ONPG) washydrolyzed by lactose-positive Neisseria. Eight strains of pharyngeal origin wereexamined. In culture reactions, seven strains resembled Neisseria meningitidis withthe exception that they produced acid from 1% (w/v) lactose. An eighth strain(V8) differed in that it did not form acid from maltose or from 1% lactose. How-ever, acid formation was observed in 10% lactose cultures of strain V8, suggestingthat entry of lactose occurred by passive diffusion, rather than as a result ofpermease activity. The enzymes which hydrolyzed ONPG were produced constitu-tively by the cells of all eight strains. Thus, specific activity in these strains was not in-creased by prior exposure to lactose, or to two other possible inducers, isopropyl-:-D-thiogalactoside or methyl-3-D-thiogalactoside. Study of cell-free extracts of one

strain showed that the enzyme was heat-labile, having a half-life of 10 min at 45 C.The enzyme was unstable at low protein concentrations, but it was protected com-pletely or partially when albumin or manganous ions were added. The enzyme ap-peared to be a typical 3-galactosidase: a-galactosides (melibiose and p-nitrophenyl-a-D-galactopyranoside) were not hydrolyzed, activity against ONPG was not de-pendent upon inorganic phosphate, and galactose was released by cleavage ofONPG. ONPG hydrolysis provided a simple and rapid method for detecting lac-tose-positive Neisseria.

The common species of Neisseria do not typi-cally form acid from lactose (Bergey's Manual).Nevertheless, lactose-positive strains have beendescribed (13, 15, 16) which bear considerableresemblance to Neisseria meningitidis. As a basisfor projected genetic and taxonomic studies ofrepresentative strains, the enzymatic action ofthese lactose-positive Neisseria on various galac-tosides was examined. Use of a chromogenicsubstrate, which has been invaluable in numerousgenetic investigations of lactose metabolism (1,12), provided a sensitive method for determining(3-galactosidase activity.

MATERIALS AND METHODSStrains. Eight strains of lactose-positive Neisseria

were generously provided by other investigators.Strains 325, 328, 329, 330, and 331, which were ob-tained in 1965 from throat cultures, were receivedfrom Daniel Ivler, Infectious Disease Laboratory,University of Southern California School of Medicine,Los Angeles. Strains A3383 and A5906, also isolatedfrom throat cultures, were received from Howard A.Fox, National Communicable Disease Center, At-

' Taken from a thesis submitted by W. P. Corbettin partial fulfillment of the requirements for the M.S.degree, Marquette University.

lanta, Ga. Strain V8 was isolated prior to 1934 froma surgically removed tonsil. This strain, which wasoriginally described by J. Jessen (13), has been pre-served at the Statens Seruminstitut, Copenhagen,Denmark, and was received from Alice Reyn, Neis-seria Department.

N. subflava, strain N15 from the collection of theUniversity of Maryland (received from M. J. Pelczar,Jr.), was examined for control purposes.The methods of preservation and propagation of

cultures have been described elsewhere (5).Media. All strains were cultivated on HIY-C agar

which consisted of Heart Infusion Broth (Difco), 0.3%(w/v) yeast extract (Difco), 0.5 mm CaCl2, and 1.0%(w/v) Ionagar No. 2 (Oxoid). The production of acidfrom carbohydrates was determined in CTA Medium(BBL). Stock solutions of carbohydrates, which hadbeen filter-sterilized with HA type membranes (Milli-pore Corp. Bedford, Mass.), were added aseptically togive final concentrations of 1 or 10% (w/v). Initialchange ofpH was encountered with some 10% solu-tions of carbohydrates and was corrected by the addi-tion of NaOH. Lactose from two suppliers (Difco andFisher Scientific Co.) was used for tests of acid produc-tion.

Plates containing 5% sucrose in HIY-C agar wereused to determine the possible conversion of sucroseto a polysaccharide which reacts with iodine (11).After incubation at 35 C for 2 days, cultures were

52

on January 15, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

LACTOSE-POSITIVE NEISSERIA

treated with drops of a solution containing 0.2%iodine and 0.4% potassium iodide (Burke's modifica-tion of Gram's iodine solution, freshly diluted 1:5with water) and were then examined for the rapiddevelopment of a blue-black color. To detect a pos-sibly weak reaction, cultures which failed to showspecific darkening with diluted solution were retestedwith undiluted iodine solution, and a control test wascarried out on the culture grown on sucrose-freeHIY-C agar.

Chemicals. All reagents were of the highest gradecommercially available: o-nitrophenyl-,3-D-galacto-pyranoside (ONPG) was obtained from SigmaChemical Co. (St. Louis, Mo.); isopropyl-I3-D-thio-galactopyranoside (IPTG), methyl-,j-D-thiogalactopy-ranoside (TMG), D-galactose, 2-(N-morpholino)-ethanesulfonic acid (MES), and Bovine PlasmaAlbumin (Pentex) were obtained from Calbiochem(Los Angeles, Calif.). Melibiose and N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (oxi-dase reagent) were obtained from Eastman OrganicChemicals (Distillation Products Industries, Roches-ter, N.Y.). Pierce Chemical Co. (Rockford, Ill.)supplied the a-D-melibiose and p-nitrophenyl-a-D-galactopyranoside. Reagent-grade sucrose, maltose,D-fructose, D-glucose, and D-xylose were obtainedfrom Fisher Scientific Co. (Pittsburgh, Pa.).

Buffers. Phosphate-buffered saline (PBS), usedroutinely, contained 0.85% (w/v) sodium chlorideand 0.05 M sodium phosphate at pH 6.7. When inor-ganic phosphate was to be excluded, 0.85% NaCl wasused with 0.05 M MES, pH 6.7.

Cell-free extracts. The cells were collected from2-liter volumes of cultures shaken for 24 hr in HIY-Cbroth, were washed once with PBS, and were thenresuspended in 35 ml ofPBS (or 0.85% NaCl solution,as indicated). A Raytheon sonic oscillator (model DF101, 10 kc, Raytheon Co., South Norwalk, Conn.),operating at maximal output for 20 min, disruptedthe cells, and the extract was freed of debris by cen-trifugation. Crystalline pancreatic deoxyribonuclease(Worthington Biochemical Corp., Freehold, N.J.;found to be free of activity against ONPG) was addedat a final concentration of 5 ,Ag/ml, and the mixturewas incubated at 28 C for 30 min. It was dialyzed(Visking dialysis tubing) against PBS or saline solu-tion (renewed periodically) at 3 C for 3 days. Thedialyzed extract was sterilized by filtration and storedat 3 C.

Assay of cell-free extracts with ONPG. j3-Galac-tosidase activity was determined by use of a modifiedversion of the procedure described by Lederberg (14).The selection ofpH 6.7 for procedures with Neisseriawas based on preliminary determinations of the pHoptimum of enzyme activity in cell-free extracts ofstrain 325. Extracts suitably diluted in PBS (includingalbumin as a stabilizing agent, whenever necessary)and ONPG solutions (10.0 mm in PBS) were equili-brated separately at 28 C. Equal volumes (2.0 ml)were mixed and, after an appropriate time, the reac-tion was stopped by addition of 1.0 ml of 1.0 Msodium carbonate. The optical density (OD) wasdetermined at 420 mA relative to a reagent blank. Thequantity of ONP liberated was estimated by compari-

son with an ONP standard curve. If a series of assayswas to be accumulated before reading, the sampleswere stored at 3 C to minimize the spontaneoushydrolysis of alkaline ONPG.The quantity of ONP liberated was found to be

linear with time, as is required for a satisfactory assay.Furthermore, a linear relationship was found betweenenzyme activity and the quantity of cell-free extract,examined in detail for strain 325.One unit of enzyme is defined as that quantity

which will liberate 1 m,mole of ONP/minute underthe conditions of assay. The specific activity is definedas the number of enzyme units per milligram ofprotein.

Assay of cell suspensions. Toluene and several alkylalcohols were tested for their ability to remove thepermeability barrier of strain 325. The greatest in-crease in j3-galactosidase activity was found with1-butanol. Used at a final concentration of 2% (v/v),it produced a three- to fivefold increase of activity,which was maximal after 10 min at 28 C. Accordingly,this concentration of 1-butanol was added to cellsuspensions of all strains prior to equilibration at 28 Cfor 15 min. After adding sodium carbonate, the cellswere removed by centrifugation at room temperature,and then the OD was determined. Other detailsduplicated those described for assay of cell-free ex-tracts.

Additional requirement for assay of strain V8.jB-Galactosidase activity in strain V8 was determinedin the presence of 0.1 M 2-mercaptoethanol. In theabsence of this agent, treatment of strain V8 witheither 1 -butanol or sonic oscillation resulted in apartial or complete loss of activity.

Assays with other substrates. Enzyme assays withp-nitrophenyl-a-D-galactoside, melibiose, lactose, andmaltose were performed as for ONPG, except thatthe liberated aglycone was measured differently. p-Ni-trophenol was determined at 400 mn. The reactionswith lactose, melibiose, and maltose were terminatedby heating to 85 C for 5 min, and the concentration ofthe liberated glucose was determined enzymaticallyby use of Glucostat (reagents and procedure suppliedby Worthington Biochemical Corp., Freehold, N.J.).

Protein determination. Gornall's biuret reagent (9)was used (the final concentration of carbonate-freeNaOH, however, was doubled). After the reaction hadproceeded at room temperature for at least 1 hr (carebeing taken to avoid trapping air bubbles after thedevelopment of viscosity), the OD was determined at360 m,u, as recommended by Goa (8), and the quantityof protein was estimated by comparison with astandard albumin curve.

Induction experiments. A slightly turbid bacterialsuspension was prepared from a 12- to 15-hr cultureon HIY-C agar, which had been subcultured re-peatedly in carbohydrate-free medium, and 1.0 mlvolumes were added to a pair of Nephelos Flasks(Bellco Glass Inc., Vineland, N.J.), which contained24.0 ml of HIY-C broth. The flasks were incubated at35 C with vigorous rotational shaking, and the ODat 650 m, was determined periodically to ascertainthat the cells were in the logarithmic growth phaseduring the test. The inducer was added to one of the

VOL. 95, 1968 53

on January 15, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

CORBETT AND CATLIN

paired cultures (final concentration of IPTG andTMG, 0.5 mM; lactose, 15.0 aiM) and an equal volumeof sterile water was added to the other. After 2-hrincubation in the presence (or absence) of inducer, thecells were collected by centrifugation in the cold,washed once with cold PBS, and then resuspended in25 ml of PBS. Corresponding cultures of Escherichiacoli B (permease-positive) and E. coli mutabile(permease-negative) served as controls. For eachculture, quantity of protein and quantity of jl-galac-tosidase activity were determined in triplicate.

RESULTS

Cultural characteristics. The eight lactose-positive strains were gram-negative diplococciwhich had the typical appearance of Neisseria.They were aerobic, catalase-positive, and oxi-dase-positive. Growth of all strains occurred onHIY-C agar at 35 C, although strains 331 andA5906 required an increased carbon dioxide ten-sion (supplied in a candle jar). To standardizeincubation, variants which overcame this CO2requirement were selected for subsequent useand were designated 331-1 and A5906-1. Isolatedcolonies of strain V8 on HIY-C agar, incubatedfor 24 hr at 35 C, were significantly smaller (0.5to 1.0 mm diameter, either with or without C02)than the colonies of the other seven strains (1.5to 2.0 mm diameter). None of the strains showedsignificant growth from diluted inocula on HIY-C agar at 28 C or at 22 C. Nutrient Agar (Difco)incubated at 22, 28, or 35 C did not supportgrowth of any of these lactose-positive strains,nor did it support growth of a group B strain ofN. meningitidis (Ne 15). Corresponding tests onN. subflava (N 15) showed growth on bothNutrient Agar and HIY-C agar at 28 C and at35 C (but not at 22 C).Seven of the strains (325, 328, 329, 330, 331,

A3383, and A5906) showed identical carbohy-drate reactions. Acid was formed from 1% con-centrations of glucose, maltose, and lactose (also10% lactose). No acid was detected in tests onsucrose, fructose, xylose, or galactose (10%galactose being negative also). Correspondingtests on N. meningitidis (Ne 15) and N. subflava(N 15) gave the same reactions, with the excep-tion of lactose tests which were negative (both1 and 10%).

Strain V8 differed in its action on two carbo-hydrates. No production of acid was observed intests on either 1 or 10% maltose. CTA mediumcontaining 1% lactose was persistently negativeduring incubation at 35 C for 6 days. In addition,a corresponding tube, heavily inoculated andincubated at 28 C, showed growth but no forma-tion of acid. However, 10% lactose was attacked,production of acid being clearly detectable afterincubation for 48 hr at 35 C.

None of the eight lactose-positive strains con-verted sucrose to a polysaccharide capable ofreacting with iodine, indicating that they lackedan amylosucrase (10). Negative tests were like-wise obtained with four strains of N. meningitidis.N. subflava (N 15), on the other hand, producedan iodine-reacting polysaccharide. Furthermore,as expected (2, 11), positive tests were obtainedwith five strains of N. perflava.

Tests of induction. Exploratory work indicatedthat ONPG was attacked promptly by cellstaken from lactose-free agar. This suggested thatthe enzyme may be produced constitutively, incontrast to the f3-galactosidase of wild-type E.coli which requires prior induction (12). The lackof inducibility was confirmed by the results ofquantitative experiments (Table 1). None of theNeisseria strains showed a significant increase inspecific activity associated with prior growth ofyoung cultures in lactose-containing broth ascompared with prior growth of young cultures inlactose-free broth. Two cultures of E. coli, usedas controls, showed the expected increase inspecific activity.

In E. coli, a product of the transgalactosi-dation of lactose (effected by the basal level ofthe f-galactosidase), rather than lactose itself,functions as the inducer (4). Therefore, we con-sidered the possibility that failure of the lactose-positive Neisseria to be induced in the presence oflactose may be due to defective transgalactosidaseactivity. This possibility was examined withstrains 325 and V8 by growing cultures in thepresence and absence of 0.5 mm IPIG andTMG. Specific activities of the Neisseria strainsdid not increase in response to these compounds,whereas both E. coli strains showed increasesgreater than those observed with lactose. Themore conspicuous increase, as expected, wasfound for E. coli mutabile (the permease-negativecontrol): IPTG produced a 100-fold increase andTMG an 18-fold increase of specific activity. Itwas therefore concluded that the enzymes ofthese lactose-positive Neisseria are constitutive.

f3-Galactosidase of strain 325. To determinewhether the enzyme of this representative lactose-positive strain was a typical ,B-galactosidase, cell-free extracts were examined. The enzyme wasfound to be unstable at elevated temperaturesand during dialysis against water. Moreover,spuriously low specific activities were found athigh dilutions of the extract. The instability wasincreasingly evident as the protein concentrationwas reduced below 100 ,g/ml. Accordingly, apreliminary study was made to determine themagnitude of the dilution effect and to find asuitable protective agent.The ,B-galactosidase of E. coli is protected at

54 J. BACTERIOL.

on January 15, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

LACTOSE-POSITIVE NEISSERIA

low protein concentrations when albumin ormanganous ions are added (17). The effects ofthese two agents, alone and in combination, onthe activity of strain 325 extracts are shown inTable 2. Albumin, added to maintain the totalprotein concentration at 200 ,g/ml, providednearly complete protection of enzyme in dilu-tions of the extract. Dilutions assayed in thepresence of manganous ions (the concentrationbeing limited by the phosphate buffer) showedincreased specific activity, although the degreeof protection was not equal to that afforded byalbumin.The possible stimulating or inhibiting effects of

TABLE 1. Specific activity offt-galactosidase afterexposure of cultures to lactose

Exposure conditionsOrganism

Lactose Lactose-free

Neisseria sp.325 .................... 33a 55a328................. 57 52329................. 121 125330................. 133 145331-1 ................. 104 99A3383................. 45 54A5906-1................ 107 138V8 ................... 26b 32b

Escherichia coli B (per- 1,060 14mease-positive)

E. coli mutabile (per- 23 12mease-negative)

a Units of ,6-galactosidase/mg of protein, usingONPG as substrate with washed suspensions ofcells previously cultured in broth containing 15.0mm lactose or in lactose-free broth.

b Assayed in the presence of 0.1 M 2-mercapto-ethanol.

TABLE 2. Effects of protein concentration andmanganous ions on f3-galactosidase activity

in a cell-free extract of strain 325

Specific activityaBacterial

ortein _Qg/sml) No addition Albuminb Mnc(2 Albumin +

(0.2 mm) Mncl,

64 180 418 434 52032 63 410 346 51016 30 403 271 491

a Units of j-galactosidase/mg of bacterial pro-tein found in assays with ONPG and added com-ponents.

b Crystalline bovine plasma albumin added at aconcentration sufficient to increase the totalprotein to 200,ug/ml.

some other divalent cations were investigated.Addition of magnesium chloride to the PBS-ONPG test system slightly increased the specificactivity; a 10% rise was found with 1.0 mm anda 5% rise with 0.1 mm. Calcium chloride, at 0.1mM, produced a 6% decrease of specific activity.No activity was detected in the presence of 0.01mM HgCl2.Two cell-free extracts of strain 325 were exam-

ined for possible dependence of ,B-galactosidaseactivity upon inorganic phosphate. One, dialyzedagainst PBS as usual, was diluted in a solutioncontaining albumin and 0.85% NaCl until thefinal concentration of inorganic phosphate wasreduced to 0.25 mm. This dilution was assayedwith ONPG in MES-NaCl buffer, and the activ-ity was found to be 43.3 units/ml. Assays per-formed in the presence of increasing concentra-tions of phosphate showed no increase of activ-ity. In fact, the activity decreased to 41.8 units/ml at a concentration of 10.2 mm phosphate.The maltose phosphorylase (7) present in thesame dilutions served as an internal control. A47% increase in activity against maltose wasfound, despite suboptimal assay conditions,when the concentration of phosphate was in-creased from 0.25 mm to 10 mm. A second cell-free extract, prepared and dialyzed in phosphate-free saline, likewise showed no dependence uponadded phosphate (maltose phosphorylase activ-ity was not detected in this preparation).

In addition, one product of the cleavage ofONPG was identified as galactose by chroma-tography on bisulfite-treated silica gel sheets(Eastman, Type K301R, Eastman ChemicalProducts, Inc., Kingsport, Tenn.). These findingsindicate that the enzyme catalyzes the hydrolysisof the substrate.The substrate specificity was examined by use

of representative a- and ,B-galactosides. Noenzymatic activity was detected in tests of a cell-free extract against two a-galactosides, melibiose,and p-nitrophenyl-a-D-galactoside, whereas thesame preparation hydrolyzed the two fl-galacto-sides tested. In sodium phosphate buffer, therate of hydrolysis of lactose was only 13% ofthat of ONPG. However, K+ has been found toactivate the f-galactosidase of E. coli when lac-tose is the substrate, whereas Na+ is the activatorwhen ONPG is used as substrate (6). In the caseof the extract of strain 325, it was found thataddition of 10.0 mm KCI to the assay systemincreased the activity against lactose by 12.6%,whereas the activity against ONPG decreased by2%. This increase occurred in spite of a highsodium ion concentration, and reduction of Na+may result in a further increase of activity againstlactose.

VOL. 95, 1968 55

on January 15, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

CORBETT AND CATLIN

The sensitivity of enzymes to heat inactivationmay have differential value. To examine thepossibility that the two ,B-galactosides may behydrolyzed by different enzymes, the cell-freeextract (325) was diluted in prewarmed (45 C)PBS containing 200 ,ug of albumin/ml. Incuba-tion was continued at 45 C for 30 min, and at 5-min intervals samples were removed and imme-diately chilled to 3 C. Each sample was assayedseparately against lactose and ONPG. The activi-ties against the two substrates decreased at equalrates over the entire period, with a half-life of 10min. The absence of different rates of inactiva-tion at 45 C supported the view that a singleenzyme was responsible for hydrolysis of bothlactose and ONPG.

DISCUSSIONAll eight strains of lactose-positive Neisseria

produced enzymes which cleaved ONPG. Weconcluded that these hydrolytic enzymes wereconstitutive, rather than inducible, as in wild-type E. coli, since activity against ONPG wasnot increased by prior growth of the bacteria inthe presence of lactose, IPTG, or TMG. Detailedexamination of cell-free extracts of one strain(325) established that a-galactosides were notattacked, activity against ONPG was not depend-ent upon inorganic phosphate, and galactosewas released by cleavage of ONPG. These find-ings indicate that the enzyme is a typical #3-galac-tosidase. Although definitive studies require theuse of a purified enzyme preparation, the viewthat lactose and ONPG are attacked by the sameenzyme was supported by the similarity of theheat inactivation curves. It is understandablethat lactose, the most probable substrate for theenzyme, should be attacked at a lower rate thanONPG when one considers that the conditionsof assay were established for ONPG, rather thanfor lactose.

Seven of the lactose-positive Neisseria showeda resemblance to N. meningitidis which is similarto that described for other strains (15). Strains325, 328, A3383, and A5906 were reactive withantiserum against N. meningitidis group B (D.Ivler and H. A. Fox, personal communications).On the basis of complement-fixation reactions,

Jessen (13) recognized that strain V8 is serologi-cally related to N. meningitidis and N. gonor-rhoeae, and recent study of V8 has shown astrongly positive reaction in the direct fluorescent-antibody test with anti-gonococcal serum (A.Reyn, personal communication). The ability toform acid from maltose and from 1% lactose isno longer demonstrable, although strain V8appears to have possessed these properties whendescribed in 1934 (13). The production of acid

in medium containing 10% (but not 1%) lactosesuggests that strain V8 now lacks a functionalsystem (permease) for active transport of lactose(entry of lactose apparently proceeds by passivediffusion). Each of the other seven strains pre-sumably possesses a permease for transport oflactose.

Information is insufficient for the taxonomistto decide whether the resemblance between theeight strains of lactose-positive Neisseria and thepathogenic species of Neisseria represents arelationship so close that these strains should beregarded as lactose-positive biotypes of N.meningitidis (or, for strain V8, of N. gonorrhoeae),or whether they represent a distinct species.Studies are needed to determine the degree ofgenetic relatedness and also to determine whetherthe strains may be associated with significantdisease.To determine the identity of a suspected mem-

ber of Neisseria, it has become standard practicein many laboratories to restrict tests of acid pro-duction to those carbohydrates which are recog-nized as having diagnostic value. Accordingly,most N. meningitidis strains isolated duringrecent years have not been examined for possibleattack of lactose. The use of ONPG for thisdiagnostic purpose may prove to be as usefulwith Neisseria as it has become with entericbacteria (3).A satisfactory qualitative test to reveal the i3-

galactosidase of Neisseria can be performed bypreparing a dense cellular suspension in 0.5 mlof a solution containing 5 mm ONPG (dissolvedin PBS, pH 6.7), and incubating this suspensionat room temperature. A positive test is indicatedby the development (usually within 1 hr) of adistinct yellow color. To exclude spontaneoushydrolysis of ONPG as a possible cause of colordevelopment, a corresponding negative controltest (for example, with N. perflava) must remaincolorless. E. coli taken from lactose agar providesa useful positive control test. The constitutivenature of these Neisseria strains suggests thatcells for the routine ONPG test may be takenfrom a lactose-free agar medium, although useof lactose (1 or 10%) agar cultures may revealan inducible type or help to identify a permease-negative type of lactose-positive Neisseria. Inview of the lability of the ,B-galactosidase of onestrain (V8), treatment of the cells with an agentto remove the permeability barrier is contraindi-cated (such treatment produces at most a five-fold increase in activity). Since most strains ofNeisseria autolyze readily, the use of a largenumber of cells and the incubation of the reac-tion mixture for 18 to 20 hr before making thefinal reading provide a sensitive test.

56 J. BACTMOL.

on January 15, 2021 by guesthttp://jb.asm

.org/D

ownloaded from

LACrOSE-POSITIVE NEISSERIA

ACKNOWLEDGMENTThis investigation was supported by Public Health

Service grant 02353 from the National Institute ofAllergy and Infectious Diseases.

LITERATURE CrrED1. BECKWITH, J. R. 1967. Regulation of the lac

operon. Science 156:597-604.2. BERGER, U. 1961. Polysaccharidbildung durch

saprophytische Neisserien. Zentr. Bakteriol.Parasitenk. Abt. I Orig. 181:345-349.

3. BULow, P. 1964. The ONPG test in diagnosticbacteriology. 2. Comparison of the ONPG testand the conventional lactose-fermentation test.Acta Pathol. Microbiol. Scand. 60:387-402.

4. BURSTEIN, C., M. COHN, A. KEPES, A.MD J. MONOD.1965. Role du lactose et de ses produits meta-boliques dans l'induction de l'operon lactosechez Escherichia coli. Biochim. Biophys. Acta95:634-639.

5. CATLIN, B. W., AMD L. S. CUNNINGHAM. 1964.Genetic transformation of Neisseria catarrhalisby deoxyribonucleate preparations having dif-ferent average base compositions. J. Gen.Microbiol. 37:341-352.

6. COHN, M., AND J. MONOD. 1951. Purification etproprietes de la ,-galactosidase (lactase) d'Es-cherichia coli. Biochim. Biophys. Acta 7:153-174.

7. FITING, C., AND M. DOUDOROFF. 1952. Phos-phorolysis of maltose by enzyme preparationsfrom Neisseria meningitidis. J. Biol. Chem. 199:153-163.

8. GOA, J. 1953. A micro biuret method for proteindetermination. Determination of total proteinin cerebrospinal fluid. Scand. J. Clin. Lab.Invest. 5:218-222.

9. GORNALL, A. G., C. J. BARDAWILL, AND M. M.DAVID. 1949. Determination of serum proteinsby means of the biuret reaction. J. Biol. Chem.177:751-766.

10. HEHRE, E. J., AND D. M. HAMILTON. 1946.Bacterial synthesis of an amylopectin-like poly-saccharide from sucrose. J. Biol. Chem. 166:777-778.

11. HEHRE, E. J., AND D. M. HAMILTON. 1948. Theconversion of sucrose to a polysaccharide ofthe starch-glycogen class by Neisseria from thepharynx. J. Bacteriol. 55:197-208.

12. JACOB, F., AND J. MONOD. 1961. Genetic regula-tory mechanisms in the synthesis of proteins.J. Mol. Biol. 3:318-356.

13. JESsEN, J. 1934. Studien uber gramnegative kok-ken. Zentr. Bakteriol. Parasitenk. Abt. I Orig.133:75-88.

14. LEDERBERG, J. 1950. The beta-D-galactosidase ofEscherichia coli, strain K-12. J. Bacteriol.60:381-392.

15. MITCHELL, M. S., D. L. RHODEN, AND E. 0.KING. 1965. Lactose-fermenting organisms re-sembling Neisseria meningitidis. J. Bacteriol.90:560.

16. MITCHELL, M. S., D. L. RHODEN, AND B. B.MARCUS. 1966. Immunofluorescence techniquesfor demonstrating bacterial pathogens associ-ated with cerebrospinal meningitis. III. Iden-tification of meningococci from the nasophar-ynx of asymptomatic carriers. Am. J.Epidemiol. 83:74-85.

17. RICKENBERG, H. V. 1959. The effect of metal ionsand proteins on the stability of the j3-galacto-sidase of Escherichia coli. Biochim. Biophys.Acta 35:122-129.

VOL. 95, 1968 57

on January 15, 2021 by guesthttp://jb.asm

.org/D

ownloaded from