Origin of Polysaccharide Depolymerase Associated with Bacteriophage Infection

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Origin of

Polysaccharide Depolymerase Associated

with

Bacteriophage Infection

PASQUALE F. BARTELL AND THOMAS E. ORR

Department ofMicrobiology,NewJerseyCollege ofMedicine andDentistry, Jersey City, New Jersey07304

Received for publication9 December 1968

Analyses, by construction of phage growth curves, indicated that the

poly-saccharide depolymerasewassynthesizedby Pseudomonasaeruginosastrains Band

BI after infection with phage 2.The kinetics of biosynthesis of the depolymerase

were foundtoparallel closelytherateofformation ofphage-directed virions, and

alterations intheexperimentalconditions of infectionwerereflected byalterations

in theproductionofenzyme.Infection with other Pseudomonasphages,84and1197,

didnotresult in thesynthesis ofdepolymerase. The enzymewasnot detectable in uninfected cultures, and no evidencewas obtained for the existence ofinhibitors

or activators ofenzyme activity in extracts of uninfected or infected cells. The results of experiments employing chloramphenicol or an auxotorphic mutant

(BI arg-) suggested that protein synthesisdenovowasessential for productionof

theenzyme. Variousmutants ofphage2

(pdp1,

pdp2), which alter the synthesisof

the polysaccharide depolymerase, have been isolated. These experimental results

stronglysupportthe role of thephagegenomein thesynthesis of thisenzyme.

Inaprevious paper,evidencewaspresented for

theexistence ofapolysaccharide depolymerasein

phage lysates of Pseudomonas aeruginosa (4).

Re-action of this enzyme with polysaccharide

ob-tained from the slimelayer of this organism

re-sulted inadecreasedviscosityandincreased levels

ofhexosamines, hexoses, andreducingsubstances.

Subsequently, the polysaccharide depolymerase

was purified 1,688-fold from crude lysates by

conventional methods of protein fractionation

(3). Maximal enzymatic activity of the purified

enzymewasfoundatpH 7.5,and,onthe basis of

gel filtration data, the molecular weight was

estimated to be 180,000. We have begun to

in-vestigate the problem of the origin of this hy-drolytic enzyme and its functional role in the virus life cycle. Evidence presented in this paper

strongly suggests that thepolysaccharide

depoly-merase, which isrecovered from crude lysates of

P.aeruginosaafter infection with phage 2, results

from de novo synthesis ofa phage-directed

pro-tein.

MATERIALS AND METHODS

Bacterial and phage strains. P. aeruginosa strainsB

andBI weredescribedpreviously(4). Isolation ofthe

auxotrophicmutantofstrain BI used in these

experi-mentsisdescribed below. Pseudomonas phages 2and

29were isolated fromlysogeniccultures of strainsB

andBI, respectively, and phages 84 and 1197 were isolated from other lysogenic cultures ofP.aeruginosa.

Media.Trypticase SoyBrothandAgar(BBL)were employedascompletemedia forbacterial cultivation and phage propagation. The minimal medium de-scribed by Freese (7) was also employed in certain experiments. It had the following composition: glucose, 5 g; disodium hydrogen phosphate, 7 g; potassium dihydrogen phosphate, 3 g; ammonium chloride, 1 g; FeCl3-6H2, 0.0003 g; CaCl122H20,

0.015 g;magnesiumsulfate,0.25g;distilledwater, 1 liter;agar (whenrequired),15 g.

Isolation ofauxotrophic mutant strain BI arg-. A modification of thedelayed-enrichment technique (9)

was employed for the isolation of a nutritionally deficient mutant of strain BI. An 18-hr culture of strain BI was centrifuged at 3,020 X g for 20 minandwaswashed andresuspendedin 0.1 M sodium phosphate-buffered saline, pH 7.5. Approximately 7 ml of thesuspension,containing 4.8X 107viable cells per ml, wasexposed to ultraviolet (UV) irradiation (Sylvania germicidallamp, G15T8) at anintensityof 120jgw/cmO. Irradiation was carried out with agita-tionof thesuspensionfor 15sec.Viablecountswere

made before and after irradiation. These conditions resulted inasurviving fraction of 0.003 of theoriginal suspension. The exposed bacterial suspension was incubatedinthe darkat roomtemperature for 2 hr, then at 37C with aeration for approximately 5 hr; 0.1-mlamounts of various dilutions were placedon sterilecellulose membranes whichwerepositionedon thesurface of minimal agarplates.After 48 hr of incu-bationat37C,thecellulose membranescarryingthe bacterial coloniesweretransferredtoplates containing complete medium, and the position of the colonies

wasmarked. Theplateswereagainincubatedat37C. 290

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POLYSACCHARIDE DEPOLYMERASE

Isolates which grew oncompletebut not onminimal medium within a 48-hr period werecharacterized as auxotrophs.Qualitative identificationofgrowthfactor requirements was made auxanographically as de-scribed by Lederberg (9).

Mutants ofphage 2. Suspensions of phage2 were treated with 1%S ethyl methanesulfonate (Eastman Organic Chemicals) in solution with 0.5 M sodium acetate and 0.001 M magnesiumsulfate (pH 7.2) as

describedbyFreese (8). At variousintervals oftime afterincubation at 37C,samples ofthismixturewere

diluted 10-fold in Trypticase Soy Broth containing 2% sodium thiosulfate and were plated onlawns of P. aeruginosa BI. Mutants were picked, plaque-puri-fied, andpropagated. Thesemutantsweredesignated pdptosignify alterationin,or absenceof, the phage polysaccharidedepolymerase,accordingtothescheme suggestedby Eisenstark(6).

Production of enzyme in phage-infected cultures. Theelaborationof phage andpolysaccharide depolym-erase by phage 2-infected bacteria was studied in phage growth experiments. Bacteriophage titrations wereperformed bysoftagar-layer, andothergeneral phage procedures were performed according to

Adams (1). Lysogenic cultures were induced by UV irradiation. Bacterialcellswerefirst grown in Trypti-caseSoy Broth to aconcentration of S X 108/ml,and then were centrifuged and resuspended in 0.1 M sodiumphosphate-buffered saline (pH7.5). Approxi-mately7mlof thebufferedsuspension, inanexposed petridish, wasirradiated withagitationat10juw/cm2

for 20sec. Afterirradiation, the bacterialsuspension was added to an equal volume of Trypticase Soy Broth (2X) and wasincubated at 37 C. In other

ex-periments, approximately 5 X 10° cells/ml, in the exponential phase of growth, were usually infected with phage at a multiplicity ofinfection (MOI) of

3 to 4. Adsorption, at 37 C for 5 min with gentle

shaking,wasfollowedbycentrifugationand resuspen-sion ofsedimented cells inanoriginalvolume of pre-warmed broth. Incubation was at 37 C in a shaker waterbath.Sampleswereobtainedatvariousintervals oftime, immediatelychilled, andcentrifugedat6,000 X gfor 10 min at4 C. Supernatantfluids were as-sayedfor free phage and enzyme. The percentageof uninfected cells was measured directly by plating samples of the culture 5 min after infection. The MOI wasdetermined bytheratio ofinput phageto

bacteria (assuming 90% adsorption) or by counting the number of surviving bacterial colony formers (assuming a Poisson distribution of input phage). Values obtained by these two methods agreed to within 15 to 20%o. The intracellular appearance of phageand enzymewas determined bythe disruption ofinfected cells. Atvarioustimes,30-mlsampleswere

withdrawn,chilled, andcentrifuged. The sedimentwas

frozen and during thawingwas ground in 0.5 ml of distilled water containing 0.5 g of alumina powder (Gamal, Fisher Scientific Co.). Extraction was

con-tinued inatotal of5ml of colddistilledwaterfor 15

min. The extracts were then centrifuged, and the supernatant fluids were assayed. Extracts of unin-fected cells were prepared in the same manner. In

certain experiments, the effect of chloramphenicol

(Calbiochem) on depolymerase synthesis was deter-mined by adding chloramphenicol (100 ,ug/ml) to phage-infected cultures at various times.

Estimation of enzyme activity. Assays were based on the release ofhexosamines from slime polysaccharide substrate aspreviously described (4). The amount of hexosamine was determined by the method of Elson andMorgan (11) as modified by Boas (5). Progress curves were constructed, and the initial velocity was determined. A unit of enzyme activity is defined as nanomoles of hexosamine released per minute per milligram of protein (or per milliliter of sample). Astandard ofD-glucosamine hydrochloride (Eastman OrganicChemicals) wasincludedin each hexosamine determination. A method of assay described by Adams and Park (2) was also useful in estimating enzymeactivity.Thedetails, asapplied tothisstudy, have been described (4).Briefly, various dilutions of enzyme were observed fortheir ability to produce a perceptibleclearingofthe bacteriallawn.

RESULTS

Infection by phage2as aspecificrequirement for the synthesis of polysaccharide depolymerase. Formation of the polysaccharide depolymerase

was routinely observed in lysates of P. aerugi-nosastrainB, which carriesphage 2 as prophage, after induction by UV irradiation (Fig. 1). Depolymerase activity was detected 90 min after irradiation and sharply increased to a maximal concentration during the following 30 to 40 min. During the same period oftime,there was almost a 3-log increase in the number of infective phage 2particles released by theinduced cells.

A similar response was observed when P. aeruginosa strain BI was infected with phage 2 (Fig. 2). Depolymerase activitywas not detected

-I0

£0

-E 10

-._

8-c

6-cn

4

-w

_

2-

0-a.0

-J

a

0

l

-000oo

w

-100o

I

a.

10

w

Ow

30 60 90 120 150

[image:2.487.250.440.437.614.2]

TIME AFTER IRRADIATION (min)

FIG. 1.Appearanceof depolymeraseand phage 2 in

culturesofstrain Bafterinduction by UVirradiation

(10,lwpercm2, 20sec).

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60 min after infection, but a signific

was present in the 90-min sample

increasing to high concentrations.

induced culture, theproduction ofde

was paralleled by a significant incr

number of infective phage, reachin

concentrations within 40 min after

appearance.

It was of interest to determine w]

phage and enzyme were first detec cellularly. The disruption of infecti various time intervals after infectic the presenceofdepolymerase and ms in the 50-min sample (Fig. 3). The

enzyme and maturephage in the

40-was further confirmed by concentr

samples 10-fold before assay. Supern

0

E

C

w

-0

a-w a

10

8

4-

2-0

0-0 0 -0

0

/

00

30 60 90 120 150

TIME AFTER INFECTION

FIG. 2. Appearanceof depolymerase a,

infectedculturesofstrain BI.

0

E

).

CK w

CLI c

w

0

10

-8

-

6-

4-

2-

0-30 50 70 90

ant amount and extracts prepared from uninfected cultures

thereafter of strain BI in the logarithmic phase ofgrowth

As in the and early in the stationary phase of growth

-polymerase were repeatedly examined and found to be

ease in the devoid of depolymerase activity. However,

ig maximal cultivation of strain B eventually resulted in

their initial detectable levelsofdepolymerase. This, of course,

could be related to the spontaneous induction

hen mature of strain B, resulting in the synthesis of both

table intra- phage and depolymerase. Experimentswere also

ed cells at conducted to determine whether phage 2

in-rn revealed fectionmight inducethe synthesis ofa substance

ature phage which, in turn, causes the activation ofa latent

absence of orrepressed enzyme. Whenextracts ofuninfected

min sample cells were mixed, in varying proportions, with

*ating these extracts ofphage 2-infected cells, no increase or

atant fluids decrease of depolymerase activity was observed.

Thus, no inhibitorof the enzyme was detectable

o in extracts of uninfected cells, and no evidence

was obtained to suggest a mechanism whereby

o0 the enzyme was released from a bound state

-1000 cr

u in extracts of uninfected cells upon incubation

- with anextract from infected cells.

F These results suggested that infection with

- 100 w phage 2 was a requirement for the synthesis of

< the polysaccharide depolymerase and that it

I

might

be a

phage-directed synthesis

de novo.

10 This

hypothesis

was

partially

corroborated

by

> thefollowingexperiments.

Infection of P. aeruginosa strains B and BI

L < by other Pseudomonas phages was studied to

0 W determine whether depolymerase synthesis was a

a specific responsetophage2infection, or whether

itmightbeamoregeneral response of these host (min) cells to phage infection. Therefore, strain BI

was infected with phage 84. Although high

con-id

phage 2 in centrations of phage 84 were produced, no

depolymerase activity was detectable (Fig. 4).

0

*1000 w E

C

*100

< w

I cn

a. <

10 W

S:--;X

_-0

lJ

>_-_lJ₀

c: w

0

10

-8

-6

-

4-2

60 90 120 150

0

-1000 cr

- 100 LLw

I

L:1

- 0 w

(min)

FiG.3. Intracellularappearanceof depolymeraseand

phage2ininfectedculturesofstrainBI.

TIME AFTER INFECTION (min)

FIG. 4. Absence of depolymerase in phage

84-infectedBIcultures. ,h

O J

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In other experiments, a lysogenic

strain BI, which carries phage 29 as

wasexposed to UV irradiation. Altk

numbers of phage 29 were syntt

detectable quantities of polysacci

polymerase were present. Similarly, i

strain B with phage 1197 failed t

synthesis of the depolymerase, alti

concentrations of phage 1197 were

(Fig. 5).

The nature of the phage 2-ass4

polymeraseactivity was characterized

studying the effects of altering the N exposing phage 2 to UV irradiation MOI was drastically reduced, there longeddelay in the appearance ofde

(Fig. 6). At an MOI of3, approximal

T

E

._

w (n

c:

w cn

>111

0 a. w 0

10

-

8-

6-

4-

2-0

-0-0

0

30 60 90 120 150

(min)

FIG. 5. Absence ofdepolymerase in phage

1197-infectedB cultures.

E 10

w

6-48

w

6

2

0

0-w

0 I

(min)

30 60 90 120 150

TIME AFTER

INFECTION

(min)

FIG. 6. Production of depolymerase in phage

2-infectedB! culturesatMO! of3.0 (0) and0.03 (0).

culture of the bacteria were infected and significant

con-s prophage, centrations of depolymerase were measured

iough great in the 90-min sample. Onthe other hand, when

iesized, no the MOI was reduced to 0.03, approximately 4

haride de- to 5%ofthe bacteria were infected and

depolym-infection of erase activity was not detected until 130 min

o result in after infection. Thus, the time ofappearance of

iough high depolymerase in infected cultures and the

quan-e produced tity of depolymerase produced appeared to be

influenced by the proportion of bacterial cells

ociated de- initiallyinfected by phage2.Suspensionsof phage

lfurther by 2wereirradiated for various periodsoftime,and

40I and of samples were used to infect cells at a ratio of

When the virus tocells of 3 to 4 (Fig. 7). With the lower

was a pro- doseofUV irradiation (5. 5% survival), the

ap--polymerase pearance of depolymerase activity was delayed

tely94% of until 120min afterinfection, and the

concentra-tion ofdepolymerase finally attained at 150min

0 was considerably lower than that of the

unir-0 radiated controls. Athigherdoses of UV

irradia-100 tion

(0.3%

phage survival),

an even more

pro-w longed delay was observed before depolymerase

E activity could bemeasured. Based on a Poisson

distribution, approximately 5% of the bacterial

100 W₀

cells

were initially infected with the phage

sus-< pension receiving the higher UV dose, whereas

a.

approximately 15%

were

initially

infected with

10

w phage exposedtothelowerUVdose.In the

con-> trol cultures, that is, phage not previously

ex-! posedtoUV,approximately95 % ofthe bacterial

oJ cellswere

initially

infected.

cr

Dependenceofdepolymerase

activity

on protein

synthesisde novo. Two typesofexperimentswere

carried out in order to establish that protein

E

0

4-

F-cn

Cl w

w -J

0

10

4-0 oo S

o A

0Ji

--I I .

60 90 120 150

TIME

AFTER INFECTION

(min)

FIG. 7. Production of depolymerase by strain B!

afterinfectionwithphage 2, UV-irradiatedto asurvival of5.5 X 102 (0) and 3.0 X 10-3 (A). Control cultures were infected with unirradiatedphage (0).

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synthesis de novo was essential for the

appear-ance of depolymerase activity. In the first

ex-periment, chloramphenicol (100 jg/ml) was

added to cultures of strain BI at the time of

infection withphage2. Theresults (Fig. 8)

indi-cated that synthesis of the depolymerase and of

the phage was completely inhibited. The

ob-served effect could not be attributed to a defect

inthe adsorptiveprocess, since more than 99%

adsorption occurred in the presenceof

chloram-phenicol. When chloramphenicol was removed

from infected cultures after a 10-min exposure,

synthesis of depolymerase and phage was

re-sumed after a slight delay. This effect was also

demonstrable when chloramphenicol was

re-movedfrom infected cultures after a more

pro-longed exposure of 30 min. In other

experi-ments, the addition of chloramphenicol was

delayed until40, 65, and 90 minafter infection.

Depolymerase activity was not detectable in

infected cultures when an inhibitory

concentra-tion of chloramphenicol was added 40 minafter

infection (Fig. 9). However, when the addition

of chloramphenicol was further delayed to 65

min, a small quantity of depolymerase was

detectable, and with greaterdelay (90 min) even

higher concentrations of depolymerase were

detected in culture fluids.

In the second type of experiment, an

auxo-trophic mutant of strain BI was infected with

phage 2 in the absence of the necessary amino

E

4-_cn

w

-J

cn

0.

(LL

w

a

10

-

8-4.

2

0

-30 60 90 120 150

TIME

AFTER

INFECTION

(min)

FIG. 8. Effect ofchioramphenicol onappearance of depolymerase in phage 2-infected BI cultures. Chlor-amphenicol (100 ,g/ml) addedat the time of

infec-tion(0); infectedcellswashedfree from

chlorampheni-colafter10 min (A);controlcultures infectedin the

absence ofchloramphenicol (0).

E

._-3

C]

w

i-J

0

a.

w

10-

8-6

0

-0

0 0

AO*--A

O::~-I I I T

30 60 90 120 150

TIME

AFTER INFECTION

(min)

FIG. 9. Appearance of depolymerase in phage

2-infected BI cultures treated with chloramphenicol at

various times after infection. Chloramphenicol (100

jug/mi) introduced at 40 mini (A), 65 min (0), and

90 min (0) after infection.

E 0 C

a..

w cn

CL

Ld

10

-

8-

4-0

a

0

0 a

I

'-o+/+/

00

30 60 90 120 150

[image:5.487.255.448.56.258.2]

TIME

AFTER INFECTION

(min)

FIG. 10. Depolymerase production by an arginine auxotroph (BI arg-) infected with phage 2 in the presence of arginine (2mg/ml; 0),in the absence ot arginine (0), andafter the addition of arginine (I) at 35 min after infection (U).

acid (Fig. 10). WhenstrainBIarg- was infected

in the absence of arginine, no depolymerase

activity was detectable throughout the 150-min

period of observation. However, when the

re-quired amino acid was added to the culture

35minafterinfection, depolymerase activitywas

detectable after a slight period of delay. The

addition of arginine to uninfected,

arginine-O

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starved bacteriahad no effect inbringing about

theappearanceofdepolymeraseactivity.

Mutants ofphage 2. Plaques of the wild-type

phage 2 are surroundedby large halos when this

phage is plated on lawns of the sensitive

indi-cator strainBI. The halo effect iscaused by the

action of the polysaccharide depolymerase,

which diffuses from the plaque area and

hy-drolyzes the slime polysaccharide of the bacteria

which surround the plaque (4). Additional

evi-dence supporting the role of the phage genome

in the synthesis of the polysaccharide

depolym-erase was obtained through the isolation of

mutantphage. Theexposureofphage2 to ethylm-ethanesulfonate resulted in the isolation of

threetypes of mutantswhichweredistinguishable

on lawns of strain BI (Fig. 11). The wild-type

phage 2 produced a turbid plaqueand was

sur-roundedbya largehalo. Aclear plaquemutant,

Cir, also produced depolymerase and was

sur-rounded by a large halo. However, the mutant

phagedesignatedaspdp1 producedaclearplaque

butwashalo-less. The absence ofapolysaccharide

depolymeraseinlysates producedbythismutant

was confirmedby enzyme tests with slime

poly-saccharide as substrate. Another mutant, phage

pdp2, also produced a clear plaque which

ap-peared to be equivalent in sizeto that ofphage

[image:6.487.43.239.369.590.2]

pdp1,but the size of the surrounding halo was

FIG. 11. Plaques produced bymutantsofphage2on

alawnofBI. Wildtype, wild, turbidplaquewithhalo;

mutantclr,clearplaquewithhalo;mutantpdp,, clear

plaque, no halo; mutant pdp2, clear plaque, reduced halo.

smallerthan theclrorwild-type halos, suggesting

an alteration in the quantity or structure of the

synthesizedenzyme.

DISCUSSION

The experimental results which have been pre-sented strongly support the hypothesis that the

polysaccharide depolymerase was synthesized

de novo as a phage-directed enzyme protein after infection.

Analyses by construction of phage growth

curves indicated that the depolymerase was

synthesized by bacterial cultures infected from

without or after induction by UV irradiation.

In both cases, the appearance of enzyme was

preceded by a period of latency during which

no enzyme was detectable. Subsequent to its

intracellular appearance at approximately 50

min, therelease ofenzymeby infectedcells was

observed to occur by 90 min after infection.

In these studies, the kinetics of biosynthesis of

the depolymerase closely paralleled the rate of

formation of phage-directed virions. Although

determinations of phage deoxyribonucleic acid

synthesis have not been made, it is likely that

the polysaccharide depolymerase is a "late"

enzyme.

The depolymerase was not detected in

unin-fected cultures. However, theenzyme and phage

were recovered after cultivation of strain B at

37 C, but thisis attributabletothe spontaneous

induction of strain B which carries phage 2

as prophage. A substrate-controlled induction

of the depolymerase was not observed, nor was

there any experimental evidence for the

exist-enceof inhibitorsoractivators ofenzymeactivity

in extracts ofuninfected or infected cells.

Infec-tion of strains B and BI by other Pseudomonas

phages, 1197 and 84, respectively, did notresult

in the synthesis of a detectable depolymerase.

The induction of strain BI, which carries phage

29 inthe prophage state, byUV irradiation also

failedtoresult in the synthesis ofdepolymerase.

Thus, theproduction ofenzyme appears to bea

highly specific response following infection of

the hostcellbyphage2andcannotbe considered

ageneral responseof thecelltophageinfection.

When experimental conditions were altered to

reduce the number of bacterial cells initially

infectedwithphage, aproportionatedelayinthe

appearance of enzyme and a decrease in the

quantity of enzyme produced, within the time

period ofthe experiments, was observed. Thus,

alterations in experimental conditions of

infec-tion werereflectedby alterations inthesynthesis

ofenzyme. The results ofexperimentsemploying

chloramphenicol or an auxotrophic mutant of

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strain BI indicated that protein synthesis de novo was essential for synthesis of the depoly-merase.

However, the present study does not exclude the possibility that somehost cell protein might be incorporated into the final active form of the enzyme. In thepresent case, in which activity is not detectable in uninfected cells, a similar mechanism could account for the appearance of enzymatic activity afterinfection. Such a mecha-nism would postulate the existence, in uninfected cells, ofa preformed specific inactive precursor. Mathews, Brown, and Cohen (10) addressed themselves to this problem inthecase of deoxy-cytidylatehydroxymethylase. Cells prelabeled by growth in the presence of methionine-methyl-'4C were infected in nonradioactive medium. The hydroxymethylase purified from these cells was virtually nonradioactive and provided direct evidence that the enzyme was synthesized de novo after infection, and that the host cell did

notcontribute any preformed enzyme precursor. However, such findings do not exclude the pos-sibility that the host cell possesses the genetic information for enzyme synthesis and that the

infection itself activates this process through

some process of derepression. In view of our experimental findings, this possibility must be considered remote in the Pseudomonas phage 2 system. Furthermore, theexposure ofphage2 to

ethyl methanesulfonate resulted in the isolation

of variousmutantswhich altered thesynthesis of

polysaccharide depolymerase and thereby

pro-vided strong support for the role of the phage genome inthe synthesis ofthis enzyme.

ACKNOWLEDG MENTS

Thisinvestigationwassupported bygrantGB7891 fromthe NationalScience FoundationandbyPublic Health ServiceGrant AI-08504 from theNational Institute of Allergy and Infectious Diseases.

LITERATURE CITED

1. Adams, M. H. 1959. Bacteriophages.Interscience Publishers, Inc., NewYork.

2. Adams, M. H., and B. H.Park. 1956. An enzyme produced by a phage host-cell system. II. The properties of the polysaccharide depolymerase. Virology 2:719-736. 3. Bartell, P.F.,G.K. H. Lam, and T. E. Orr.1968. Purification

andpropertiesofpolysaccharide depolymerase associated with phage-infected Pseudomonas aeruginosa. J. Biol. Chem.243:2077-2080.

4. Bartell, P.F.,T.E. Orr, and G.K. H. Lam. 1966. Polysac-charide depolymerase associated withbacteriophage infec-tion.J. Bacteriol. 92:56-62.

5. Boas, N. F. 1953. Methodforthedetermination of hexosa-minesintissues. J. Biol. Chem. 204:553-563.

6. Eisenstark, A. 1967. Bacteriophage techniques, 449-524.In K. Maramorosch and H. Koprowski (ed.), Methodsin virology, vol. 1. Academic Press, NewYork.

7. Freese, E. 1959.Thespecificmutagenic effectof baseanalogues onphage T4.J. Mol.Biol. 1:87-105.

8. Freese, E.1963. Induced and spontaneousmutations in bac-teriophage, p. 3-18.In W. J.Burdette (ed.), Methodology inbasic genetics. Holden-Day, Inc.,Publisher, San Fran-cisco.

9. Lederberg, J. 1950. Isolation and characterization of bio-chemicalmutantsofbacteria. MethodsMed.Res.3:5-22. 10. Mathews, C. K., F.Brown, and S. S. Cohen. 1964. Virus inducedacquisition ofmetabolic function. VII.

Biosynthe-sisde )?ovOofdeoxycytidylate hydroxymethylase. J. Biol. Chem.239:2957-2963.

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