T1 Genes Which Affect Transduction

0022-538X/80/03-1122/07$02.00/0

Ti

Genes Which Affect Transduction

L. D. BORCHERTANDH. DREXLER*

Departmentof Microbiology and Immunology, Bowman Gray School of Medicine of Wake Forest

University,

Winston-Salem,

NorthCarolina27103

Amber mutants of

Ti

were grown on each of three donor strains which were

identicalexceptthatthey carried different suppressors: respectively, supD, supE,

andsupB. The efficiency with which the mutants were able to transduce was

tested aftergrowthoneachdonor. Ingeneral, it was found that functions which

control the synthesisof phage DNA usually causedsignificant increases in the

efficiency of transduction (EOT). Afewmutants located in genes essential for

headproduction caused significantdecreases in EOT. The presence of a particular suppressorinadonorcan causenoteworthy changes in the EOT by certain of the mutantphages. Amber mutations ingene 3 of

Ti

wereextremely sensitive to the

particular suppressor presentin the donor, showing a 17-fold decrease in EOT

compared with othermutants after growthin donorswith the supD suppressor

anda 75-fold increase after growthin supEdonors. Increases in EOT by early genesof

Ti

donotseem tobe causedbyalack of competitionof bacterial DNA

withphageDNAduring packaging since,in mostinstances, infective phage were

produced inrelatively normalamounts comparedwith wild-type

Ti.

Phage DNA

synthesis and degradations of the host chromosome are closely coupled in

Ti

infections;we believe that increasesin EOT by mutants ofearlyfunctions are

duetoinefficientdegradation of the hostchromosome.

The virulent coliphage

Ti

is a generalized transducing phage

capable

of

transducing

either bacterial markers (6) orprophages suchasA or

Mu (3, 7). It has been shown in

previous

work that thepresence of mutations in certaingenes orsites of the chromosome ofEscherichia coli donor cells can alter the

efficiency

with which

Ti

transduces. For

example,

deletion of theesp

site resultsinareduced

efficiency

of

transduc-tion

(EOT)

of the biotinoperon(7), whereas

Ti

lysatespreparedondonors withrecBmutations transduce

prophage

X less

efficiently

than

lysates

preparedonrecB+ donors

(8).

Incontrast tothe effects ofespdeletionsorrecB

mutations,

stimn-ulation of the

products

of therecEsystemof E. coliortheredsystemof

prophage

X

during

the

production

of

Ti

lysates

increasesEOT of

pro-phage X DNAby

Ti (8).

Others have

reported

that alterations in EOT by

phage

P22 can be causedby bacterialmutations (5).

Phagemutationswhich affectEOTby phage

P22 (15) and phage

Pi

(18) have also been

reported. Likewise, phage mutations which in-crease EOT by

Ti

have been observed and mapped in gene 2.5

(M.

D. Roberts and H.

Drexler,

unpublished observations).

Inthis re-port, the effects which amber(am) mutationsof

Ti

genes haveonEOT are

reported.

The pur-pose of the experiments was to determine whether mutations inmostof the known genes

of

Ti

could be

rapidly

screenedforaneffecton

transduction. The

principle

ofthe experiments is that insomeof thesuppressorcells the prod-uctof thesuppressed amber mutations would be altered instructure andfunction and therefore

cause a

significant

alteration in the ratio of

trans-ducing

particles

toPFU.

Thus,

wehoped tobe able to identify genes whose products play a

significant rolein

modifying

theEOT of bacte-rial markers by

Ti.

The EOT of X PFU by a

variety of Tlammutantsgrown,respectively,on a series of three strains of E. coli which are

nearly isogenic but contain different amber sup-pressors wasassayed; each suppressor addeda

different amino acid when it

suppressed

an am

mutation. On theone

hand,

itwasobserved that

suppressed

ammutations located in

phage

genes

whose functions have been characterized as

being

essential in the normal

synthesis

of

Ti

DNA (early

functions)

consistently led to in-creases in EOT thatwere

significantly

greater than average.Onthe other

hand,

suppression

of ammutations inseveral

Ti

genes whose

prod-ucts areessentialforhead formation

consistently

led to low EOT values. A few mutant

phages

showed large shiftsin EOT that

depended

on

the particular suppressorpresent in the donor

cells.

(Someof theresults inthisreportwere sub-mitted in

partial fulfillment

of the

requirements

ofWake Forest

University

for theM.S.

degree

byL.D.B.)

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MATERIALS AND METHODS

Bacterialandphagestrains. The donor strains of E. coli and the recipient SA216 used in these experimentsare described inFig. 1. The variousam mutants of Ti that were used are indicated on the mapof Ti inFig.2.E. coliW3350tonA is resistantto Ti (6).

Media andlysate preparation. The standard me-dia have beendescribed inapreviousreport.Likewise, lysate preparation was by standard methods previ-ouslyreported (6, 13).

Assay of X PFU formed by Ti transduction. When Tlam mutants are plated on the Su- strain SA216, theefficiency ofplatingisreduced compared withplatingon anSu'indicator(e.g.,S1654). Presum-ably the Ti plaques formed by Tlam mutants on SA216aredueto revertantphages,and the number of suchrevertantsvariesfromtwo tofive ordersof mag-nitudeless than the total PFU measuredon anSu' indicator. IfaTlammutantisplated with the mixed indicator system of SA216 and W3350tonA, the re-vertant Ti plaquesare turbid duetoovergrowth by theTl-resistant strainW3350tonA.

Lysates ofTi prepared, for example, on the Su' donor strain S1652

(Ximm'dg)

are not capable of giving risetoAPFUonnonlysogenic indicators such W3350 orW3350tonA. However, a Tlam lysate pre-paredonS1652(Ximm434dg)willproduce A PFU when plated with the SA216 indicator. Presumably A PFU canarisewhenaTitransducing particle formed dur-ing growth of Tlam on a Su' donor lysogenic for

Aimm'dgpackaged some, or all, of the Aimm4'dg

prophage DNA and injected it into SA216. If

Aimm'dg

DNAwasinjectedintoSA216and

subse-quently recombined with the residual DNA of the deleted, AimmA prophage which was resident inSA216,

afullycompetentAimm4' phage would be produced

Aimm 434 j

SU+ gal Nel SRA Mgal b

Donor UP' SB'

Su- cCl * J* bio

Recipient

FIG. 1. SU' (i.e., suppressor-containing) E. coli strains used.S1652, S1654, and S1656 were supplied to us by S.Adhya (1) and lysogenized by us with

Aimm4dg;

thisAimm434dg(supplied by A. Campbell)

retainstheAMbut not theALfunction.S1652carries the supD suppressor, which reads the UAG codon andadds serine to the growingpolypeptide chain. S1654 hassupE, which reads UAG and adds gluta-mine. S1656has supB, which reads UAA or UAG andpossiblyaddslysine(2).S1652,S1654, andS1656

areisogenic except for the different suppressors. The Su (does not suppress amber mutations) E. coli strainSA216 was provided by A.Campbell and pre-viously called R954(Acry)(4). Thedeletion in SA216 removes the Nfunction of A and penetrates the bac-terialchromosome beyond the galactose operon.

In-fornationabout Aphages may befound in reference 12.

PHENTYPE DOW HD T DA DA TAIL HEAD

GENE 2 2.5 3 3.5 4 5 6 7 6 9 10 11 e 13 14 15 16 Ir 1I

MU1ANT 16 3 tar 6 201 23 15I 15 1 13 2937 1045 11 4 730

I0 1 1 13 111 19 l2I5

FIG. 2. Variation of the Ti map of Figurski and Christensen(9). Mutantsdesignated by numeralsare amber mutants; each number represents an inde-pendently isolatedmutantwhich isnotidentical to others located in thesamegene. Numbers 201 and 221 wereisolated andmapped byD. A. Ritchie and D. T. M. Martin (unpublished observations). The remaining ambersare thoseofMichalke andwere mapped by Michalke(14)andby Figurskiand Chris-tensen (9). The tar mutant (Roberts andDrexler, unpublishedobservations)wasnotused in thisreport but is mentioned in thetextand is addedfor com-pleteness. The meanings of thephenotypic designa-tionsare asfollows:DO,nosynthesisofphage DNA; HD,meansabsenceofhost DNAdegradation; Tor TAIL, noproductionofcomponenttails; DA,arrest (i.e.,earlyshutoff) ofphageDNA synthesis; HEAD, noproduction of competent heads.

(see Fig. 1). In the absence ofspecific immunity

re-pressor, Aimm= would produce aninfective center andaA-typeplaquewould be formedontheindicator cells. Figure3showsanexperimental plateonwhich a Tlam grown on an Su' strain lysogenic for

Ximm'dg

was plated with SA216 andW3350tonA.

Lambda PFU were clearly distinguishable from Ti PFU even when the one plaque type was superim-posedonthe other(Fig.3). To avoidproblemsofviral interference orexclusion which might arise ifa cell wereinfected with bothaTi andaAimm3 genome (10), the ratio ofTi PFU (Ti titer obtainedonSu' cells)toSA216 cells(i.e.,themultiplicityofinfection)

neverexceeded0.2. SincenoAPFUcanbeproduced by the system exceptthroughtransduction,theEOT is simply the ratio of the concentration of A PFU (assayedonSA216plus W3350tonA)tothe concentra-tionofTiPFU(assayedonSu' cells).

For eachTlammutant used, atleasttwo lysates

weremadeoneach of the three donorcells. The EOT by Ti grownon aparticular donor isanaveragevalue obtainedbyperformingatleasttwoexperiments with each lysate. In other words, eachEOT value is the averageofaminimumof fourexperiments.

Derivationof median EOT values. All the Tlam mutants inourcollection of35 wereableto produc-tivelyinfectS1654

(Aimm"dg).

Mostof the Tlam (33 of35) couldproductively infect S1652 (AimM434dg),

and some (12 of 35) wereable tomultiply onS1656 (Aimm43adg).For technicalreasons wewereunable to obtainareproducible EOT value forwild-typeTithat couldbeusedas astandardtocompareall the EOT values.Therefore, for eachsetof EOT values obtained with aparticular donor, wederived a median EOT value with which the EOT value of each individual Tlam couldbecompared. For11 ofthe 19Ti genes tested,wehadonlyasingle ammutation. However, 33,

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2 3 354 5 6 7 8 9 11 1213 1415 16 1718

TI GENES

FIG. 3. Plating ofTlammutants.Lysates for given Tlam mutants were prepared on an Su' E. coli

(kimm434dg) donor and, assayplatesweremade by the usualagaroverlay technique,with both the Su-recipientsE. coliSA216and E. coli W3350tonA as

indicators. ComparedwithX,Tlam+revertantsform

relatively large plaques and,since Ti is unable to lyse tonA cells, the plaques are turbid. Lambda

plaquesareformed afterAimm434dgDNA,which has

been transferredto SA216recipients by Ti,

recom-bines withthe defectiveAimmAprophage presentin

SA216 toform a Ximm434 infective center. Lambda

plaques arecharacteristically smaller than thoseof Ti and, since Ximm434 can lyse both SA216 and

W335OtonA, relativelyclearer.

forsomegenes wehadasmany assixnonidenticalam

mutations. Topreventtheresults obtainedwith

sev-eralmutantsinasinglegenefromundulyinfluencing the value ofthe median EOT, only oneEOT value

from each gene (i.e., the value nearest the median)

wasusedtoderive themedian EOT. RESULTS

Transduction by Tlam mutants grown

on different suppressor strains. Lysates of Tlammutantswereassayed for theirabilityto transduce DNA of

Ximm434dg,

the indicatorof transduction being the formation of a X-like

plaque (see Materials and Methods). For each of therespectivedonors, theEOT ofindividual Tlam mutantswascomparedwith themedian

EOT value for thatparticular donor. The ordi-natein each ofFig.4through6 isthelogarithm of the ratio ofEOT values for individual mutants to the median EOT; the abscissa is the gene

number of the 19 Ti genes for which amber mutationswereavailable. The advantageof

us-ing suchaplotis that n-fold differences inthe ratio of individualEOT values and the median

EOTare

equidistant-from

the referenceratio of

FIG. 4. EOT values of Tlam mutants, using at

least two independently produced lysates of each

Tlam mutant prepared on the E. coli S1652

(Aimm434dg) donor strain which carries the amber suppressorsupD.Atleasttwo separateEOT values were experimentally derived for each lysate. Each

pointonthegraph is,therefore,basedontheaverage

ofatleastfourassaysofEOT byagivenTlamstrain.

Eachpoint is thelogarithm ofthe ratio ofan average

EOTvalue ofagiven ambermutant (identified by

numberonthegraph)to themedian EOT(see

Ma-terialsand Methodsforthe calculation ofthe median

EOT). The horizontal lineon the graphrepresents

the median EOT reference value of1. Forpoints

above the median, the number usedto identify an

ambermutation isabove the point;for points below themedian, theidentifyingnumber is below thepoint.

MedianEOT is 9x 10-7.

1 regardless of whether the differences are

greater or less than 1. Forexample, afivefold

increase in the EOT ofaparticularmutant

com-pared with the median would leadtoaratio of

5, whereas a fivefolddecrease would lead toa

ratio of0.2; the logarithms of 5 and 0.2,

respec-tively, are equidistant from the reference ratio

of 1;thus, the effects of individual mutationscan

bedirectly compared regardless of whether they

causeincreasesordecreases in the EOT.

We believe thatafivefold difference from the

medianEOT identifiesamutation which

signif-icantly affects transduction. The fivefold value for the identification ofsignificant changes in transduction was chosenfor the following rea-sons. More than 320 EOT valueswereobtained

while assaying the transduction ability of the lysates of individualmutants.We found that less than 10% of the EOTvalues ofanygivenmutant

grownon a particular donor varied from each

otherbyasmuchasthreefold and less than 5%

by fivefold, even whenassays of different (i.e.,

10=

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TI GENES

FIG. 5. EOT valuesofTlammutants, using trans-ducing lysates obtained by growth of individual

Tlammutantsonthe donor E. coli S1654(Aimm434dg)

strain whichcarries the ambersuppressorsupE.For

otherdetails,seethelegendtoFig.4. MedianEOT is4x10-7.

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TI GENES

FIG. 6. EOTvaluesofTlammutants,using

trans-ducing lysates obtained by growth of individual Tlammutantsonthedonor E. coliS1656

(Aimm4dg)

strainwhich carries theochresuppressorsupB. For otherdetails, seethelegendtoFig.4.MedianEOT is6x10-8.

individually prepared) lysates were compared.

Thereforeconsistent fivefold differences in EOT valuesarenotapttooccurby chance.

Further-more,useof theStudentttest tocompareEOT

values ofindividual mutants with the median showedthatthree-tofourfold deviationshada

t value of 5 to 0.5%; fivefold deviations were

always less than 0.5%.

Thedatacollected during thetransduction of A PFU by avariety of Tlam strains grown on

differentdonors(Fig. 4-6)canbesummarizedas

follows.Atotal of22EOT valueswereobtained

byusing Ti strainscontaining amber mutations

in genes 1, 2, 3.5, and 4 (early genes whose

products control the synthesis ofphage DNA).

It wasobserved that16of the 22 EOTs obtained for the phage strains which had amutation in

an early gene transduced significantly higher

(some as muchas 50-foldhigher) than the me-dian EOT. A total of41 assays for EOT were made withphagescontaininganamber mutation in genes whose

products

are essential for tail formation (gene 3 plusgenes 5

through 11).

Of the41 assaysof EOT

by

the different tail mu-tants,7 were atleast fivefold above the median. The Tlam6 mutation in gene 3 showed a

re-markable shift in EOT whengrownondifferent donors[a 17-fold decrease whengrownonS1652

(Ximm434dg)

anda75-fold increase whengrown

onS1654

(Ximm434dg)].

Of the17 assaysof EOT byphage strains with mutations ingeneswhose products were essential for head formation (genes12

through 18),

none wasfivefoldgreater

than the median.

However,

4 of the 17 EOTs

were at least fivefold less than the

median,

whereas

only

1EOTassayoutof63

using early

and tailmutantswas

significantly

less thanthe medianEOTs.

Insummary, thereseemedtobeapatternin

the effectthat

suppressed

ambermutations had

on the EOT

compared

with the median EOT. Mutantslocated in geneswhich control

phage

DNA

synthesis

tended to transduce at

signifi-cantly

higher

levels,

whereas

significant

reduc-tion in the EOT seemed to be due

chiefly

to

head mutations. A few tail mutations caused significant increases in the

EOT,

whereas one

tail mutation

(am6

in gene

3)

caused

signifi-cantly lower or higher EOTs,

depending

on

whichsuppressor was presentin thedonor. Average burst size. The average burst size

ofsomeof thephage strainswasmeasured. The

phage strains selected for testing

formed

a

cross-section of mutant strains which: (i) contained

earlygenedefects which tendedto transduce at greaterthanaverage rates(i.e.,

Tlaml6,

Tlam5, and Tlam2l); (ii) were mutated in tail genes

whichtransducedatratessignificantlydifferent

from the median EOTs (i.e., Tlam6, Tlam4l,

and

Tlaml8);

(iii) hadEOTsnearly identical to

the median for all three donors (i.e., Tlaml3 andTlam29);or(iv) transducedatEOTs signif-icantly less than themedian EOTs (i.e., Tlam4,

Tlam7,and

Tlam30).

The results(Table 1) showed wide variations in the average burst size from phage strain to

phagestrain or with a given phage strain from donor to donor. Usingthe average burst size of

wildtype as areference point, it can be seen that

onlyonemutant strain[Tlaml6 grown onS1652

(Ximm434dg)]

had an average burst size

signifi-I

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TABLE 1. Average burst sizes of certainTiam mutants ondonorstrainsa

Avg burst size on:

Ti Amber

mu-gene tation S1652 S1654 S1656

(Ximm4"dg) (Aimm4"dg) (Ximm4.Udg)

- Wild type 59 63 74

1 16 94 60 10

2 5 27 46

21 54 60

3 6 53 11

41 38 9

6 18 7 44 3

9 13 28 8 8

11 29 50 10 10

16 4 9 8

17 7 53 56 9

18 30 51 28

aIndividual donor strains were infected with

Ti

at a mul-tiplicity of 0.4 to1.After a 5-min adsorption at 35°C, the infectedcellsweredilutedtogive less than one infectedcell

per tubeand incubated at35°Cfor 60 min. Thetotal contents ofeach tube wereplated with anSu'indicator and counted next day. Using the Poisson distribution to determine the numberof bursts, the average burst size was calculated as the total number ofphage, less thesupernatantphage, divided by the total number of bursts determined to have occurred in the volumeassayed. Each value in the table is the average of at least two separate experiments in which 20 to 50 bursts were counted.

cantly

greaterthan that of the wildtype.Certain

of the

phage

strains showed decreases in the

averageburstsize of2-to20-foldcompared with wild-type

Ti.

For the most part, the average

burst size seemed tobeunrelated tothe EOT.

For

example,

Tlaml3 and Tlam29 had EOTs

whichwereaboutthesame asthemedianEOTs

no matterwhichof the three donor strainswas

usedtopropagatethe

phages;

however, the av-erage burst size varied from 28 to 50,

respec-tively, on S1652

(Ximm43dg)

and 8 to 10 on

S1654

(Aimm434dg)

and S1656

(Aimm434dg).

For

Tlaml6,

thesevenfold decrease inaverageburst

size onS1656

(Ximm434dg)

couldreflect a

scar-city of

phage

DNA

during

maturation and thus explain the sevenfold increase in the EOT.

How-ever, the

relatively large

increases in EOT

by

Tlam5 orTlam2l

grown

onS1652

(Ximm434dg)

and S1654

(Ximm4 dg)

or T1am16 grown on

S1654

(Ximm434dg)

(see

Fig.

4and

5)

were not

accompanied

byany

significant

decrease in the

averageburst size.A

comparison

ofthe average

burstsizes of the other

phages

with theEOTs revealed no changes in the burst sizes which couldexplain changesinthe EOT thatweredue to ascarcityor anoverabundanceof

phage

DNA

duringmaturation. For

example,

Tlam6 hada

burstsizeof7whengrownonS1652

(Aimm434dg)

(Table 1) and anEOT

significantly

lower than the median

(Fig. 4);

therefore,

the lowEOT of Tlam6grownonS1652

(Aimm434dg)

cannotbe

ascribed to an overabundance of

phage

DNA

duringmaturation which caused an increase in average burst size and thus a lower ratio of transducingparticles to PFU.

Average latent period of phage strains

andthe growthrateof the donor cells. We

tested the average latent period of the phage

strainslistedin Table 1 andfound no significant

differences in the lengths ofthe periods (data not presented). We measuredthe growth rate of

all three donor strains during exponential

growth(the conditions of lysateformation) and

found no significant differences in growthrate

(datanotpresented).

DISCUSSION

Theeffect ofamber mutations on the EOT by

phage

Ti

wastested with mutationslocated in 19of the20knowngenesof

Ti.

Wherever

pos-sible, individual Tlam strains were grown,

re-spectively, on three donor strains which are

nearly isogenic but contain different suppres-sors. Of the35nonidentical Tlam strains avail-able to us, 12 were able topropagate on

all

3

donors and21 could propagate on 2 of the

do-nors. Regardless of the particular suppressor present inthedonor,mostmutations locatedin

phage genesknown tocontrol the synthesis of phage DNA (genes 1, 2, 3.5, and 4; the early genes) causedasignificant (i.e., fivefold) increase

intheEOT compared with the median EOT.A

mutation inagenewhose

product

isessential for

tail

production

(i.e.,

am6in gene 3) showedan

EOT which was significantly lower than the medianEOTwhenTlam6wasgrown onS1652

(Ximm434dg)

and

significantly

higher than the

median after

propagation

onS1654

(Aimm434dg);

thiswasthe most

noteworthy shift

inEOT

as-sociated with

growth

ondonorsdiffering onlyin

thesuppressorpresentinthe donor. Mutations in four of the seven genes whose

products

are

knowntobeessential for head formation

trans-duced

significantly

less

efficiently

than the

me-dian after

propagation

on at least one of the three donor strains.

The same

recipient

strain

(SA216)

wasused

in

all

experiments,

and the same indicator of

transduction wasused

(formation

ofa

Ximm434

PFU) to score transduction.

Therefore,

we

as-sume that

significant

variation between the

EOTs of the different Tlam strains grown on

thesamedonormustbe caused

by

alterationsin

proteinfunctioncaused

by

the insertionof the

specific amino acid

specified by

the

particular

suppressorpresent in thedonor.We believe that

in

general,

variation in thenumberof

transduc-ing particles

produced

arises from one of the

following

reasons:

(i)

an alterationin the

avail-abilityofphageDNA

during

maturation;

(ii)

an

alteration in the

availability

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Ti

during maturation; or (iii) an alteration in the specificity of packaging DNA. Several examples based onprevious work will serve to illustrate

ourmeaning. (i)Ti degrades the bacterial chro-mosome (9). Therefore, less and less bacterial DNA is available for packaging into mature particlesasmaturationprogresses.Asaresult of

degradation of bacterial DNA, premature lysis hasarelativelygreatereffectondecreasing the

availability of phage DNAcompared with bac-terial DNA(13).(ii) Ti phagewithmutationsin

gene 2.5 do not degrade the bacterial

chromo-some.Therefore, bacterialDNA isavailablefor packaging during the entire maturation period. The mutants of gene 2.5 (called tar mutants becausetheytransduceatanalteredrate)show

asignificant increase in the EOT compared with

tar'

strains (Roberts and Drexler,unpublished observations). (iii) Phage P22 and Ti package phage DNA by similar mechanisms (11, 17). The substrate forpackagingisashortconcatemerof

phage DNA. Packaging beginsataspecific site

in the phage DNA and proceeds sequentially (11, 17). Presumably the packaging ofbacterial DNA by P22 or Ti isinitiated at sitesonthe

bacterial chromosome which resemble the

spe-cificinitiation sites of thephage DNA (5, 7, 15, 16). The HT (high-transducing) mutants of phage P22 arelessspecific than HT+ phages in

theinitiation ofpackaging either phageor

bac-terial DNA(16). The HT mutation of phage P22 leadsto relativelymore packaging ofbacterial

DNA with respect tophage DNA and thus an

increase in theEOT(15).

Theresultspresented in Fig. 4, 5, and 6 illus-trateagreatdealofvariability in EOT fromone

Tlamstrain to another. We think it would be futile and nonrewarding to attempt to explain

everychange. Rather,wewilldiscussonlythose

changes whichoccurwithsomeconsistency (so as to reveal a trend) or which by their very

naturearestriking. After growthonatleastone

of the three donors, nearly every one of the

strains whose ambermutation is located inone

ofthe earlygeneshas anEOT which is

signifi-cantly higher than the median. The data

pre-sented inFig. 4, 5, and 6 showed that 16 of22 measurementsofEOTsby mutations ingenes1, 2, 3.5, and4were significantly higher (5-to 50-foldhigher, depending onthe donor) than the

medianEOTs. Therefore, weconclude that

al-terations ingeneproducts whichcontrol phage

DNAsynthesis haveatendancytoincrease the ratioof transducing particlestoPFU. Notmany of the observed increases in EOT caused by mutations inearlygenescanbe explainedby the

simplest ofexplanations, namely, thatanaltered

product of the suppressed mutation causes a scarcity of phage DNA. An examination ofthe

averageburst sizes of

suppressed

mutations in

the

early

genes

(Table 1)

shows that the number

ofphages

produced

compares

favorably

to the number

produced by

wild-type

Ti.

Only

in the

case of

Tiaml6

grown on S1656

(Aimm434dg)

wasit

possible

to conclude thatan increase in

EOT

probably

resulted from adecrease in the

production ofPFU (see

Results).

In

theory,

low EOTs might resultfromconditions which lead

to a larger than normal burst size.

However,

none of the mutants whichtransduced

signifi-cantlyless

efficiently

than the medianEOTgave

higher than normal bursts

(compare

EOTs in

Fig.4, 5,and6andaverageburstsizesinTable

1ofTlam6,

Tlam4,

Tlam7,

and

Tlam30).

Webelieve that the best

explanation

for the increased EOT

by

suppressed

amber mutations

inearlygenes

probably

lies inthe close

coupling

of hostDNA

degradation

and

phage

DNA

syn-thesis discovered

by

Figurski

and Christensen

(9). In other words, the

proteins

produced by

the suppressed mutations do not make

phage

DNA less available but rather make bacterial

DNAavailableover alongerperiod of time due

to a less efficient breakdown of the bacterial chromosome. In the absence of evidence indi-catingthat

suppressed

mutationsof headgenes

diluteouttransducing

particles by

production

of

an overabundance ofPFU, we are ata loss to

explain why

headmutationsareabletocause a

significant

reductioninEOT. A

possible

expla-nation is that the mutations cause a delay in

head maturation to a

point

where most of the bacterial DNA has been destroyed before the

packaging

ofDNA begins. In our opinion, the

most interesting mutant studied was the am6

mutation in gene 3. The

large

shift in EOT causedby

growing

Tlam6 indifferent

suppres-sors[low after growthonS1652

(Aimm434dg)

and highonS1654

(Ximm434dg)]

leads us to suspect

that the product of gene 3 plays a significant roleindetermining the ratio of transducing

par-ticles. This idea is strengthened by the obser-vation that the EOT ofam41 in gene 3 is also greatly dependent on the donor used to make the lysate. The product of gene 3 has been identifiedas being essential for

Ti

tail

produc-tion(9). However,gene 3is, intriguingly, flanked by genes 2.5 (host DNA degradation) and 3.5

(early shutoff of phageDNAsynthesis;seeFig. 2). Since the remaining genes of

Ti

show the

functional

organization typical of phages (see

Fig. 2), it ispossible thattheproduct of gene 3 mayplayarole in thesynthesis or processing of DNA.

The data in this report have enabled us to

identifyanumberof genes whose products are able to causesignificant alterations in the ratio oftransducing particles to PFU produced by

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1128

infectedcells.By using donorcellswith different suppressors, wehave been ableto examine the

effect ofinserting differentaminoacids into gene products. Since the suppression of the various

amber mutations abolishes the lethaleffects of

the mutations, the changes we have observed must be due to changes in the activity of the

suppressed gene products. At present we are

unabletodetermine whether thechangescaused

by thesuppressedproteins have anindirect

ef-fectontheEOT (by

making

phageorbacterial

DNA more orless available) or a direct effect

onthe ability of bacterialrelative tothe phage

DNAtobepackaged by phage heads.Wehave found thatcertaintypesofgenestendtocause

certain characteristic changes in EOT. This work increases ourunderstanding of transduc-tion. The methods usedinthisreportprovidea

screening method for therapid identification of

geneproducts which affect transduction and,as

inthecaseofgene 3of

Ti,

permitone toidentify

geneswhichare

likely

to

play

an

important

role

inthe formation oftransducing particles.

ACKNOWLEDGMENTS

This workwassupported byPublic Health Service grant no.AI07107 from theNational Institute ofAllergyand Infec-tious Diseases andby grantno.PCM77-26639 from the Na-tionalScience Foundation.

We thankTonya Reavis for her expert technical assistance. We also thank S. Adhyafor supplying uswith the strains which weadapted foruse asdonors.

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