414 MICROBIOLOGY: SCHINDLER AND SCHUHARDT PROC. N. A. S.

3Wilt, F. H., Biochem. Biophys. Res. Commun., 11, 447 (1963).7Brachet, J., et al., Biochim. Biophys. Acta, 72, 662 (1963).8 Gross, P. R., and G. H. Cousineau, Exptl. Cell Res., in press.I Hultin, T., Exptl. Cell Res., 25, 405 (1961).10 Wilt, F. H., and T. Hultin, Biochem. Biophys. Res. Commun., 9, 313 (1962).11 Melton, C. R., Genetics, 48, 901 (1963).12Tyler, A., Am. Zoologist, 3, 109 (1963).13 Gross, P. R., W. Spindel, and G. H. Cousineau, Biocheem. Biophys. Res. Commun., 13, 405

(1963).14 Cavanaugh, G., ed., Formulae and Methods of the Marine Biological Laboratory, IV (Woods

Hole, 1956).15 Herberg, R., Science, 128, 199 (1958).16Bray, G., Anal. Biochem., 1, 279 (1960).17 Lowry, O., et al., J. Biol. Chem., 193, 265 (1951).18 Scherrer, K., and J. Darnell, Biochem. Biophys. Res. Commun., 7, 486 (1962).19 Fraenkel-Conrat, H., B. Singer, and A. Tsugita, Virology, 14, 54 (1961).20 Hurwitz, J., et al., these PROCEEDINGS, 48, 1222 (1962).21 Reich, E., I. H. Goldberg, and M. Rabinowitz, Nature, 196, 743 (1962).22 Levinthal, C., A. Keynan, and A. Higa, these PROCEEDINGS, 48, 1631 (1962).23 Monier, R., et al., J. Mol. Biol., 5, 311 (1962).24 Nemer, M., and S. G. Bard, Science, 140, 664 (1963).25 Maggio, R., and C. Catalano, in preparation.26 Hultin, T., Experientia, 7, 410 (1961).27 Gross, P. R., and G. H. Cousineau, J. Cell Biol., 19, 260 (1963).

LYSOSTAPHIN: A NEW BACTERIOLYTIC AGENT FORTHE STAPHYLOCOCCUS

BY C. A. SCHINDLER* AND V. T. SCHUHARDT

DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF TEXAS, AUSTIN

Communicated by Wilson S. Stone, January 16, 1964

Interest in bacteriolytic agents of microbial origin has been manifest sinceNicolle' reported that filtrates of Bacillus subtilis had a lytic effect on "B. de Shiga,B. typhique and B. charbonneux." Fleming2 extended this interest to bacteriolyticagents from other biological sources by describing the bacteriolytic enzyme, lyso-zyme. Numerous investigators have utilized this enzyme to enrich our knowledgeof cell wall and plasma membrane composition, and cell diffusion kinetics.3Many of the extracellular bacteriolytic substances have been derived from cul-

ture filtrates of the family, Actinomycetaceae.4 5 Extracellular bacteriolytic sub-stances also have been reported from such diverse genera as Diplococcus,6 Micro-coccus,7 Streptococcus,8 Bacillus,9' 10 Flavobacterium,", 12 and Staphylococcus.13' 14The literature dealing with bacteriolytic substances active against Staphylococcus

pyogenes (aureus) has been reviewed by Elek. 15 In addition to extracellular staphy-lolytic agents, the production of autolysins (endolysins) by staphylococci has beenreported. 16-20 A unique staphylococcal endolysin, virolysin, has been described.21. 22Production of this endolysin requires induction by certain staphylococcal bacterio-phages, and is only active against killed or stressed staphylococci. In addition to

Dow

nloa

ded

by g

uest

on

Apr

il 4,

202

0

VOL. 51, 1964 MICROBIOLOGY: SCHINDLER AND SCHUHARDT 415

exo- and endolysins, bacteriophages must be considered whenever new bacteriolyticphenomena are encountered.

During a series of attempted transduction studies with staphylococci, a smallwhite colony surrounded by an area of growth inhibition was observed in theStaphylococcus aureus lawn. The isolate was given the code designation K-6-WI.It proved to be a gram-positive coccus with a specific antagonistic activity againststrains of the genus Staphylococcus. The present paper deals with the characteris-tics of this microorganism and its spectrum of antagonistic action.

Methods.-Media: Unless otherwise stated, the media, reagents, and techniques used inidentification of the K-6-WI isolate were those described by Shaw, Stitt, and Cowan.23 Theorganism was maintained, and surface growth characteristics were determined, on Trypticase-soy(Ts) broth (Baltimore Biological Laboratories, Baltimore, Md.) at pH 7.2-7.3 containing 2%(w/v) added agar (Difco, Detroit, Mich.). Gram staining characteristics were determined on18-hr Ts broth and agar slant cultures. All agar surface antagonism experiments were performedby cross-streaking 18-hr Ts broth cultures of the test organisms on Ts agar plates.

Harvesting staphylolytic filtrates: Staphylolytic filtrates were recovered from Ts broth culturesof K-6-WI after incubation at 37 in 1-liter Fernbach or fermentation flasks on a reciprocatingshaker operating with a 2.5-in stroke at a rate of 76 cycles/min. Preliminary studies indicated amaximum yield of staphylolytic substance when the pH, after an initial decrease from 7.3 to ca.6.5, increased to pH 7.5. This usually occurred at 11-27 hr, depending upon both the size of theinoculum and quantity of growth medium, and was used as a determinant of harvest time. Longerincubation resulted in higher pH and lower yields. The cultures were pooled and centrifuged at40 for 20 min at 13,200 g to sediment the cells. The supernates were filtered through cellulosemembranes (Schleicher and Schuell, Bac-T-Flex, 0.5 ,upore diam, Scientific Products, Evanston,Ill.) and were stored at -20°. Hereafter, the staphylolytic factor will be referred to as lyso-staphin.

Quantitative assay of lysostaphin: A rapid and reproducible assay was devised to compare theactivity of different harvests of lysostaphin and to assess the contemplated purification procedures.The assay organism chosen was Staphylococcus aureus FDA 209P. The cells in an 18-hr, Tsbroth, shake culture were sedimented at 13,DO g and washed twice with 0.05 M tris(hydroxy-methyl)aminomethane-HCl (Tris) buffer at pH 7.5, containing 0.145 M NaCl (buffered saline).After adjusting the Klett Summerson photoelectric colorimeter (Klett) containing a #54 filter(500-570 m,) to give a reading of 110 with 0.001 M barium sulfate, the washed S. aureus cells wereresuspended in buffered saline to give a Klett reading of 300. The addition of 1.5 ml of this S.aureus suspension to Klett tubes, containing sufficient buffered saline (with or without lysostaphin)to yield a total volume of 5 ml, gave initial Klett readings of 125 ± 5. The test and control prep-arations were incubated in a 370 waterbath, and Klett readings were made at 5-min intervals.One unit of lysostaphin was designated as being contained in that amount of test material whichgave a 50% reduction in turbidity of the standard S. aureus suspension in 10 min at 37°. Byconstructing a standard curve, the unit value of any lysostaphin preparation could be establishedwith a minimum of time and effort.

Cell wall and soluble cell substance (SCS) preparations: A concentrated suspension of Staphylo-coccus aureus FDA 209P cells was washed and resuspended in 0.145 M NaCl solution. Separateportions of these cells were used (1) unheated, (2) heated at 1210 for 30 min, and (3) unheatedand subjected to disintegration by no. 13 Ballotini beads in a Mickle Tissue Homogenizer (M.T.H.)operated at 40 for 50 min. The latter was used for cell-wall preparation. The disintegrated cellwalls were washed and recovered as described elsewhere24 25 and were suspended in bufferedsaline.To obtain SCS, 18-hr Ts broth cultures of K-6-WI were centrifuged, washed twice, and re-

suspended in Tris buffer. Separate portions of this concentrated cell suspension were subjected todisintegration in the M.T.H. operated at 40 for 20 min, and to ultrasonic vibration for 50 min in awater-cooled, 9-kc magnetostriction oscillator (Raytheon Mfg. Co., Waltham, Mass.). Afterremoval of glass beads by filtration through coarse sintered glass filters, the residual intact cellsand cellular debris were sedimented by centrifugation at 13,200 g for 20 min at 4°.

Dow

nloa

ded

by g

uest

on

Apr

il 4,

202

0

416 MICROBIOLOGY: SCHINDLER AND SCHUHARDT PROC. N. A. S.

Results.-(l) Characteristics of K-6-WI: Colonies of K-6-WI grown on Ts agarplates for 18 hr were 2-3 mm in diameter, opaque, white, circular, flattened convex,with a smooth glistening surface, entire edge, and a butyrous consistency. Stainedpreparations from 18-hr Ts broth and agar cultures revealed gram-positive cocci,1.0-1.2 .L in diameter, dispersed singly, in pairs, short chains, tetrads, and as smallclusters.Under aerobic conditions, the organism produced acid from glucose, sucrose,

lactose, D-mannose, and D(+) galactose. It failed to produce acid from mannitol,D(+) xylose, maltose, salicin, and glycerol. Using the anaerobic technique ofHugh and Leifson,26 acid was produced with the fermentation of glucose. Themethyl red and the Voges-Proskauer tests were negative. Ammonia was produced,and nitrates were reduced to nitrites. Indole was not produced, and starch wasnot hydrolyzed. Gelatin liquefaction by the Frazier27 method was positive. Acidwas produced in litmus milk, resulting in its coagulation. K-6-WI could not utilizeammonium dihydrogen phosphate as a sole source of nitrogen. Good growth ofK-6-WI was obtained on Ts agar and in Ts broth containing 12 per cent (w/v)NaCl.The K-6-WI organism produced catalase, but failed to produce coagulase. No

hemolysins were produced for rabbit, sheep, or human erythrocytes. The organismwas not susceptible to any of 26 Blair staphylococcal typing phages.

(2) The bacteriolytic spectrum of lysostaphin: The first indication that the anti-bacterial agent of the K-6-WI culture was actually bacteriolytic in nature wasobtained when K-6-WI and various strains of Staphylococcus aureus were cross-streaked on Ts agar plates. Not only was the S. aureus growth adjacent to theK-6-WI culture inhibited, but with continued incubation the area of clearing ofthe S. aureus growth was extended (Fig. la and b). This could have been theconsequence of staphylococcal endolysins coming into play after death due to apurely lethal action of the K-6-WI agent. To resolve this possibility, heat-killed(650) S. aureus cells were suspended in liquefied Ts agar, and overlays (ca. 2-indiam) were made on Ts agar plates. When the K-6-WI culture was streakedacross the turbid agar overlay and incubated, a zone of decreased optical opacity,which increased in width with increased time of incubation, was observed adjacentto the K-6-WI growth.

Fifty-four viable strains of Staphylococcus aureus have been tested for suscepti-bility to lysostaphin by the cross streak and/or the tube method. Included werehemolytic (a and ,3) and nonhemolytic strains, coagulase positive and negativestrains, 12 representative Blair bacteriophage propagating strains, 19 bacteriophagetype 80/81 hospital strains, 11 antibiotic-resistant strains, and 2 mouse virulentstrains. Also tested were 5 Staphylococcus epidermidis (saprophyticus) isolates,4 of which produced penicillinase. All 59 of these Staphylococcus spp. cultures wereattacked by the K-6-WI lytic factor. The S. epidermidis strains were lysed at amarkedly slower rate than the S. aureus strains.The bacteriolytic spectrum of lysostaphin against other bacterial species was

determined using washed suspensions of viable cells in buffered saline. Klettreadings on test and control (no lysostaphin) preparations were used to establishevidence of lysis. Staphylococcus aureus FDA 209P cell suspensions were includedto be certain of the lytic activity of the lysostaphin used, The 27 nonstaphylococcal

Dow

nloa

ded

by g

uest

on

Apr

il 4,

202

0

VOL. 51, 1964 MICROBIOLOGY: SCHINDLER AND SCHUHARDT 417

FIG. 1.-(a)S. aureus cross-streaked withmicroorganism

- ~~~K-6-WI after 16-a la + i _hr incubation.

(b) Same as(a) after 40-hrincubation.

(c) S. aureussuspension after10-min incuba-

a b tion withoutIn.~lysostaphin ( X

1400).at - i _ _ *E b 0* .. AS.(d) S. aureussuspension after

r be,a, .x*< ... ', > * 10-min incuba-dj'W_*XK$-it8tion with 0.2

14'~* ml lIysostaphinlo- %f ast~v $ ; * E A w (X 1400).- o ^ b R (Courtesy ofat̂̂r'at;.4<',4 S'~,, o the Armed

Forces Instituteof, Pathology,Washington 25,

i*Fa;+>>$_4 _ .-^ * 5.;^9<D. C.)

C d

species not lysed by lysostaphin are as follows: Micrococcus lysodeikticus, Bacillussubtilis, Bacillus megaterium, Escherichia coli, Aerobacter aerogenes, Proteus morganii,Proteus rettgeri, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens,Lactobacillus plantarum, Gaffkya tetragena, Sarcina lutea, Corynebacterium diphtheriae(gravis), C. diphtheriae (mitis), Streptococcus lactis, Streptococcus fecalis, Strepto-coccus pyogenes, Bordetella pertussis, Brucella abortus, Klebsiella pneumoniae, Pas-teurella pestis, Pasteurella tularensis, Salmonella paratyphi, Salmonella schottmuelleri,Salmonella typhosa, Vibrio cholerae (Inaba), V. cholerae (Ogawa), and Lysteriamonocytogenes (type 1). (Micrococcus lysodeikticus and Bacillus megateriuminclude cell walls and heat-killed cells; Escherichia coli includes heat-killed cells.)Additional tests on B. subtilis, B. megaterium, S. lutea, S. marcescens, P. aeruginosa,A. aerogenes, and G. tetragena indicated that the lysostaphin was neither bacterioci-dal nor bacteriostatic for these organisms.

(3) Lytic activity of lysostaphin against viable, heat-killed (1210) cells, and cell-wall fragments: Staphylococcus aureus cells were examined microscopically beforeand at intervals after the addition of a small quantity of lysostaphin. Samples(0.01 ml) of the S. aureus FDA 209P suspension were spread over circular areas(1 cm in diam) on a single, large microslide and stained by the gram method.Shortly after exposure to lysostaphin there was a decrease both in numbers andin the number of clusters of S. aureus cells. As the time of interaction betweenlysostaphin and cells increased, the remaining intact cells had less affinity for thegram stain (Fig. Ic and d). With passage of time, considerable gram-negativecell detritus appeared in the smears, and eventually all morphologically intact cellsdisappeared. Figure 2 illustrates the rapid decrease in viable S. aureus cells cor-related with decreasing optical density of a test suspension.

Dow

nloa

ded

by g

uest

on

Apr

il 4,

202

0

418 MICROBIOLOGY: SCHINDLER AND SCHUHARDT PROC. N. A. S.

90

sr-801

70

Slog .nube, of su..oIs a /

col O of S. )atscelrase0yho0lysostiph70 ce w ls io20 > 0 ; 0 l'

TIME~ ~ ~~IN40TS O a 2 5 3

othecalddnitio (ofea0.2 iomlG3-ctoofl-02m lysostaphin;(o)viablecelawtnKdW solulatcellv

8sotaphin to a Staphylococcus substance, and viable cell control; (a) autoclaved cell control;aureus cell suspension. (-) cell wall control.

The results presented in Figure 3 are not comparable on a substrate concentra-tion basis since the Staphylococcus aureus suspension used for the cell-wall experi-ment wras considerably more concentrated than were those used in the associatedviable and autoclaved cell preparations. The viable-cell reduction of turbiditycurve is typical of the results obtained with all preparations containing viable S.aureus cells and lysostaphin. The autoclaved S. aureus cells gave an initial rateof reduction of turbidity at least equal to that for viable cells. However, after 5mmn the apparent lysis of the autoclaved cells decreased rapidly and attained onlyapproximately 50 per cent of that observed for viable cells. This incompletereduction of turbidity of the autoclaved cells was probably due to the presence ofinsoluble, coagulated proteins resulting from the heat treatment.28 S. aureus cellskilled at lower temperatures and exposed to lysostaphin have given turbidimetricreadings approaching that of viable cells. The apparent initial rate of lysis andthe extent of lysis of the S. aureus cell-wall preparation were considerably less thanthat observed for viable cells, which may be a consequence of the greater substrateconcentration and an incomplete attack by the lytic agent on cell walls. WhenK-6-WJ SCS, prepared by either method of cell disintegration, was substituted forthe lysostaphin, no lysis of the S. aureus cells occurred.

Figure 4 records the results of an experiment designed to determine whether theactivity of lysostaphin was modified as a consequence of the lytic reaction. Forthis experiment, Staphylococcus aureus FDA 209P cells were concentrated to thepoint that 0.2 ml in a total volume of 5 ml gave a Klett reading of 125. For thetest preparation, a Klett tube received 4.55 ml of buffered saline, 0.2 ml of theconcentrated S. aureus cell suspension, and 0.25 ml of the lysostaphin. The testpreparation and a control without lysostaphin were incubated at 370, and Klettreadings were recorded at 5-mmn intervals for 15 min, at which time ca. 80 per centreduction in turbidity was observed. After an additional 15 mmn at 370, a second0.2-ml sample of the concentrated S. aureus ceells was added to the test preparation,

Dow

nloa

ded

by g

uest

on

Apr

il 4,

202

0

VOL. 51, 1964 MICROBIOLOGY: SCHINDLER AND SCHUHARDT 419

90r

>. 70 28

504024'~~~~ ~ ~~~~~~~~~~~~~~~~~~/l ysstaphin + cells in serum

0 40 >/a ysostaphin + cells in buffered soline200-v cells in serum without lysostaphin

0 -

20 100

TIME IN MINUTES

FIG. 4.-Sustained lytic activity 80of lysostaphin on viable Staphylococ-cusaureus cells. ( 0) 0.25 ml of lyso- 40staphin added to primary cells; (Q) 4 0 20 30 40 50 60 702nd addition of cells; (o) 3rd addi- TIME IN MINUTEStion of cells; (A) 4th addition of FIG. 5.-Activity of 0.2 ml of lysostaphincells; (-) 5th addition of cells; (0) on Staphylococcus aureus cells in the presence1.0 ml lysostaphin added to compa- of pooled human serum.rable cells; (V) 0.20 ml lysostaphinadded to comparable cells; (a) cellcontrol.

and the incubation and Klett readings were repeated. This procedure was repeateduntil 5 separate 0.2-ml samples of cells had been exposed to the original 0.25 ml oflysostaphin. No significant difference in either the rate or extent of lysis of thefirst 4 cell samples was observed in this experiment. A slight decrease in the rateof lysis was observed with the fifth cell sample. This decrease may have been dueto one or a combination of the following factors: (a) the increased viscosity of thepreparation, resulting from the liberated cell components, (b) a slight inactivationdue to the prolonged incubation, or (c) the volume change of the test preparationeffected by the repeated addition of cells. When 1 ml of lysostaphin was added toan identical S. aureus preparation, a significantly greater rate of lysis was observed,indicating that the prior results were not the consequence of an excess of lysostaphinin the original 0.25-ml test preparation. Also, the fact that the fifth S. aureus celladdition to the original 0.25 ml of lysostaphin was lysed at a significantly greaterrate than the cells of another identical cell preparation exposed to 0.2 ml of thematerial, indicated that more than 80 per cent of the original lytic capacity waspresent after lysis of 4 aliquants of S. aureus cells.

Since serum inhibits certain bacteriolytic reactions, the lytic action of lysostaphinon Staphylococcus aureus cells suspended in human serum was compared withthat of an equivalent amount of cells suspended in buffered saline. Just prior toeach Klett reading the serum preparations received an equal volume of bufferedsaline, and the saline preparations received an equal volume of serum to compen-sate for the optical density of the serum. Figure 5 illustrates the results of thisexperiment which indicate that in the presence of human serum the rate of lysis of S.aureus cells by lysostaphin was reduced but the total lysis within 65 min was equiv-alent to that noted in the buffered saline preparation. This lag may be com-parable to that noted for the fifth S. aureus sample in Figure 4, wherein the in-creased viscosity of the preparation appeared to be one possible explanation forthe slightly decreased rate of lysis.

Dow

nloa

ded

by g

uest

on

Apr

il 4,

202

0

420 MICROBIOLOGY: SCHINDLER AND SCHUHARDT PROC. N. A. S.

Discussion.-The gram-positive coccus (K-6-WI) was isolated from the lawn of aStaphylococcus aureus strain which had been exposed to the Blair typing phage no.6. Based upon morphology, biochemical tests, and the organism's tolerance tohigh salt concentration, it is identified as a member of the genus Staphylococcus.The unique characteristic of this organism is its ability to produce a potent bac-teriolytic agent, which has been proved active against more than 50 strains ofstaphylococci and inactive against 27 species of 19 other genera.The possibility that the K-6-WI lytic factor might be a bacteriophage is elimi-

nated by the following observations: (a) the rapidity of lysis is greater than thatcaused by virulent bacteriophage; (b) the lytic factor is active against all of awide variety of phage types of Staphylococcus aureus as well as 5 strains of S.epidermidis; and (c) there is no appreciable change in the lytic capacity of S.aureus preparations subsequent to their lysis. The latter also excluded lysis fromwithout by an avirulent bacteriophage.The possibility that lysostaphin is a lysozyme of microbial origin is excluded.

It does not share the same antibacterial spectrum of action with lysozyme, eitherfor gram-positive organisms (i.e., Micrococcus lysodeikticus) or gram-negative organ-isms rendered susceptible to lysozyme by heat treatment29 and although lysozymeattacks certain strains of staphylococci, only coagulase negative strains are suscepti-ble.30 Lysostaphin is active against all strains of Staphylococcus aureus tested re-gardless of coagulase production or other known strain differences.

In a subsequent paper, we will present procedures used in the partial purifica-tion and concentration of lysostaphin with additional characteristics of this material.Also preliminary results on the lysostaphin treatment of mice infected with Staphy-lococcus aureus will be reported.Summary.-A gram-positive coccus (code designation K-6-WI), assigned to the

genus Staphylococcus, has been shown to produce extracellularly a lytic factor activeagainst all of 59 other staphylococcus strains tested. The K-6-WI lytic factorlysostaphin, was inactive against both viable and heat-killed K-6-WI cells and cellsof 27 other species, many of which have been reported to be attacked by previouslydescribed bacteriolytic agents of microbial origin. Lysostaphin lysed both viableand heat-killed S. aureus cells. It is a new antibacterial agent for the staphylococci.A rapid assay for quantitating lysostaphin has been devised.

* Present address: Armed Forces Institute of Pathology, Washington 25, D. C.1 Nicolle, M., Ann. Inst. Pasteur, 21, 613 (1907).2 Fleming, A., Proc. Roy. Soc. Biol., 93, 306 (1922).3 Salton, M. R. J., J. Gen. Microbiol., 18, 481 (1958).4 Waksman, S. A., Microbial Antagonism and Antibiotic Substances (New York: The Common-

wealth Fund, 1947), 2nd ed.5 Welsch, M., J. Gen. Microbiol., 18, 491 (1958).6 Avery, 0. T., and G. E. Cullen, J. Exptl. Med., 38, 199 (1923).7 Meyer, K., J. W. Palmer, R. Thompson, and D. Khorazo, J. Biol. Chem., 113, 479 (1936).8 Hirsch, A., and D. Wheater, Nature, 168, 607 (1951).9 Greenberg, R. A., and H. 0. Halvorson, J. Bact., 69, 45 (1955).10 Strange, R. E., and F. A. Dark, J. Gen. Microbiol., 16, 236 (1957).11 Kotani, S., T. Hirano, T. Kitaura, K. Kato, and T. Matsubara, Biken's J., 2, 143 (1959).12Kotani, S., K. Kato, T. Matsubara, T. Hirano, and M. Higashigawa, Biken's J., 2, 211

(1959).13 Kashiba, S., K. Niizu, S. Tanaka, H. Nozu, and T. Amano, Biken's J., 2 50 (1959),

Dow

nloa

ded

by g

uest

on

Apr

il 4,

202

0

VOL. 51, 1964 BIOCHEMISTRY: WIBERG AND BUCHANAN 421

14 Richmond, M., Biochim. Biophys. Acta, 31, 564 (1959).15 Elek, S. D., Staphylococcus Pyogenes and Its Relation to Disease (Edinburgh and London: E.

and S. Livingstone, Ltd., 1959).6 Bronfenbrenner, I., and R. F. Muckenfuss, J. Exptl. Med., 45, 887 (1927).17Salmon, J., Compt. Rend. Soc. Biol. (Paris), 143, 717 (1949).'8Welsch, M., Compt. Rend. Soc. Biol. (Paris), 143, 721 (1949).19 Welsch, M., and J. Salmon, J. Gen. Microbiol., 3, xxvii (1949).20Welsch, M., and J. Salmon, Compt. Rend. Soc. Biol. (Paris), 144, 1929 (1950).21 Ralston, D. J., M. Lieberman, B. Baer, and A. P. Krueger, J. Gen. Physiol., 40, 791 (1957).22 Ralston, D. J., B. Baer, M. Lieberman, and A. P. Krueger, J. Gen. Microbiol., 24, 313 (1961).23 Shaw, C., J. M. Stitt, and S. T. Cowan, J. Gen. Microbiol., 5, 1010 (1951).24 Salton, M. R. J., and R. W. Horne, Biochim. Biophys. Acta, 7, 177 (1951).25 Newton, B. A., J. Gen. Microbiol., 12, 226 (1955).28Hugh, R., and E. Leifson, J. Bacteriol., 66, 24 (1953).27 Frazier, W. C., J. Infect. Diseases, 39, 302 (1926).28 Salton, M. R. J., Bacteriol. Rev., 21, 82 (1957).29 Warren, G. H., I. Gray, and P. Bartell, J. Bacteriol., 70, 614 (1955).30 Kern, R. A., M. J. Kingkade, S. F. Kern, and 0. K. Behrens, J. Bacteriol., 61, 171 (1951).

STUDIES ON LABILE DEOXYCYTIDYLATE HYDROXYMETHYLASESFROM ESCHERICHIA COLI B INFECTED WITH

TEMPERATURE-SENSITIVE MUTANTS OF BACTERIOPHAGE T4*

BY JOHN S. WIBERGt AND JOHN M. BUCHANAN

DIVISION OF BIOCHEMISTRY, DEPARTMENT OF BIOLOGY, MASSACHUSETTS INSTITUTE OF TECHNOLOGY

Communicated January 20, 1964

The present studies were undertaken as an attempt to demonstrate altered prop-erties in the enzyme, dCMP1 hydroxymethylase, formed on infection of E. coliB by genetically altered bacteriophage T4. An earlier report from this laboratory2demonstrated that this enzyme is undetectable in extracts of E. coli B infected witha T4 mutant, am N122;2 the site of mutation of this strain has been located bygenetic mapping in gene 42.4 Among the temperature-sensitive (ts) mutants ofT4 isolated by Edgar and Lielausis5 are some that map in this same gene. ThedCMP hydroxymethylases formed in E. coli B by two of these, ts G25W and tsL13, have been studied in some detail. The results demonstrate that each of thetwo mutant enzymes is more temperature-sensitive than the wild type, and that thesensitivity of one differs qualitatively from that of the other. An interesting prop-erty of the wild-type enzyme is reported, namely, its ability under certain conditionsto regain most of its activity after heat inactivation at 400C.

Methods and Materials.-The sources of the following materials were: folic acid (C grade), d-cytidine, and dCMP, California Corp. for Biochemical Research; C'4-labeled formaldehyde, NewEngland Nuclear Corp. and Volk Radiochemical Co.; 2-mercaptoethanol (Eastman grade) andthe disodium salt of EDTA, Eastman Kodak Co.; Tris, Sigma Chemical Co. The preparation ofdHMP has been described.2 Reagent-grade hydroxylamine hydrochloride was obtained fromBaker Chemical Co.The method of isolation of the ts mutants has been described.5 T4D, a revertant' of am N82

(gene 44),4 was used as the wild-type phage and is referred to as T4ts+. Mutant ts A41 was in-cluded in most experiments as a second reference phage; this mutant maps genetically in cistron3914 the same cistron in which am N1162. 4maps, and presumably contains the genetic information

Dow

nloa

ded

by g

uest

on

Apr

il 4,

202

0