Evidence for a new intermediate state of the viral chromosome during cooperative infection by host-modified Lambda phage

VIROLOQY

24, 71-83

Evidence

for

(1964)

a New

During

intermediate

Cooperative

State infection

lambda HERBERT

WEINFELD

of the

Viral

Chromosome

by Host-Modified

Phage’ AND

KENNETH

PAIGEN

In the X (C/K) system of host-controlled variation, the cooperative infection of restricting cells by host-modified phage occurs at high multiplicity of infection (m.o.i.). This was prevented by an initial exposure of the infected complexes to an anaerobic environment for 3 minutes. Subsequent exposure to air did not restore cooperative infection. If the infected complexes were initially formed under aerobic conditions for approximately 3 minutes they were no longer sensitive to anaerobiosis. The occasional infection that occurs when host-modified h is added to a restricting population at multiplicities less than 1 was insensitive t.o a brief period of anaerobiosis, as were all infections that did not involve host-controlled variation. These results dist,inguish the two possible modes of infection of restricting cell populations by host-modified phage: overcoming the restriction barrier by cooperation at high m.o.i.; and the infection at low m.o.i. of rare cells that are unable to restrict. The indifference to anaerobiosis which develops in the first 3 minutes suggests the early conversion of the incoming h.C chromosome to a new form. The yield of snccessful infections obtained after sequential infection by host-modified phages, the results of marker rescue from modified phage by unmodified phage, and the recombination data obtained in crosses between genetically marked modified phages indicate that this initial event in cooperative infection is t,he conversion of h,C to a new form of the h chromosome (h.C,). The new type of chromosome is distin~ished from X.C by its indifference to aIlaerobiosis, and from X.K by its behavior during marker rescue and its dependence upon a cooperative process for subsequent replication. After sequential infection of Escherichia coli K by h.C it was found that the secondary phage not onlg underwent cooperation with the primary phage and increased the number of cells yielding that phage, but unexpectedly acted to suppress the average burst size of the primary phage. It is concluded from the recombination data that cooperation represents a physiological complementation among the essential functions required to initiate phage replication. No evidence was obtained for the loss of any appreciable amount of genetic material from host-modified phage in successfully infected cells.

X. C, successfully infect strain IX of E. co& (i.e., produce progeny) with very low efficiency (Bertani and Weigle, 1953). This low efficiency was considered (Paigen and Weinfeld, 1963) to arise as a consequence of the independent restriction by strain I< of a number of essential sites in the x .C

INTRODUCTION

Phage X undergoes a host-induced modification when propagated in Escherichia coli strain C. The phages which arise, termed 1 Supported by a research from the United States Public

grant Health

(AI-03027) Service. 71

72

WEINFELD

chromosome. When by chance at least one copy of each of these sites escapes from restriction, phage replication can proceed. Thus, the severe restriction of X. C in strain K cells is overcome in an increasing fraction of the infected population as the average number of phages entering the cells increases. This effect has been termed cooperative infection. Those successful infections which result when only a single X. C chromosome enters a K cell can be attributed to the presence in K cell populations of rare exceptional cells that are unable to restrict X-C. The experiments reported here concern two aspects of infection by host-modified X: the first of these is whether a direct distinction can made between the two modes of infection which occur, on the one hand from the existence of exceptional cells, and on the other from cooperative infection. It was found that the latter, but not the former, of these two modes is quite sensitive to anaerobiosis. The second aspect concerns the early events occurring during cooperative infection. It is concluded that the first step in cooperative infection by X. C is its conversion to a new intermediate form of the X chromosome, designated 1. C,, and that the multiplicity-dependent step is a subsequent process, analogous to complementation, which occurs between X. C, chromosomes. MATERIALS

AND

METHODS

Terminology. The terminology used is that described previously (Paigen and Weinfeld, 1963). To identify a phage prepared in a given bacterial strain, the name of the strain follows that of the phage; thus X+ + . C is wild-type X propagated in strain C, and kh. K is a double mutant propagated in strain K. A restricted phage is one whose probability of initiating a successful infection is low by virtue of a host-induced modification. The cell which produces the restricted phage is termed the modifying host; the cell in which its efficiency of plating is low is the restricting host. Bacterial strains. The strains of E. coli K were the galactose nonfermentor gal l-2- (W-3350) and its parent gal+ (W-3110),

AND

PAIGEN

both obtained from Dr. J. J. Weigle. The E. coli strain used as the modifying host was the C gal- mutant described earlier (Paigen and Weinfeld, 1963). Saturated broth cultures of E. coli CR 63/X sensitive X+h and Xch and E. coli C600 sensitive to all unrestricted phages of the present study were used as indicators in recombination experiments. Bacteriophage stocks and techniques. Unless stated otherwise, phage and bacterial strains and the techniques and media used for their preparation and titration were as described previously (Weigle et al., 1959; Paigen and Weinfeld, 1963). Infection conditions. Since the results of infection by host-modified X are quite sensitive to the precise circumstances under which infection occurs, the relevant experimental conditions are described in some detail. Cells to be infected were grown in log phase for at least three generations in fresh, aerated tryptone broth to a titer of approximately 2 X 10s. The cultures were then chilled and centrifuged in the cold. The resulting pellets were resuspended at approximately 2 X log cells/ml in fresh ice-cold broth containing 1O-2 or 1O-3 M MgS04, which had been previously aerated vigorously for 30 minutes while ice cold. Dilutions were made in this medium to provide the desired cell concentrations. Infection was carried out in 0.5- to 1.0 ml-volumes in 13 X loo-mm test tubes at 37°C. For some experiments the adsorption mixture was gassed. In such cases, the cell suspensions were pregassed in the cold for at least 5 minutes and transferred to 37” without interruption of gas flow. In some of the experiments phage was present during this cold pregassing period; in others the phage was added after bringing the cultures to 37”. Phage adsorption was terminated by icing the tubes and adding 2.0 ml of ice-cold 0.01 M ,MgSO,. Appropriate dilutions were then made into cold 0.01 M MgS04. Infective centers were determined by plating aliquots on fresh, saturated broth cultures of the appropriate indicator bacteria. That almost all the resulting plaques arose from infected complexes, not from free phage, was indicated by the fact that

COOPERATIVE

INFECTION

treatment of identical aliquots with chloroform destroyed nearly all infective centers. Recombination experiments. In experiments where Mz.C and its corresponding wild-type X++ .C were allowed to infect E. coli K (W-3350), the infected cells were plated on a mixed indicator consisting of 1% E. coli CR 63/X in E. coli CBOO. Parental type plaques were scored as clear or as uniformly turbid, respectively. Cells which yielded mixed bursts were detected as plaques which consisted of a smooth turbid center, surrounded by a sharp clear ring, and as mottled plaques. For the measurement of recombination frequencies, the infected cells were diluted to a concentration of 3 to 6 X lo4 cells/ml in broth and the mixture was aerated at 37” for 2 hours to permit lysis. To obviate phenotypic mixing, the progeny phages were adsorbed to E. coli K (W-3350) at 1 X log cells/ml in broth containing 0.01 M MgS04 for 11-12 minutes at 37” and aliquots were plated on E. coli CR 63/X and on the mixed indicator. Recombination frequencies were not calculated as the percentage of recombinants among the total yield of progeny. Because of the phenomenon of suppression, which is described subsequently, one parent of a cross between restricted phages emerges in considerable excess over the other. A statistical correction based upon a population mating theory, such as that of Visconti and Delbriick (1953), is not applicable to this case since not all cells yielding the majority parental type also yield the minority parent or recombinants. Under these circumstances, the safest measure of recombination frequencies was judged to be the ratio of recombinant to minority parental type phages among the progeny. In crosses between Xch and X+ +, where Xch was the minority parent, this was taken as the ratio of x+h (turbid) plaques to Xch (clear) plaques on CR 63/X after removal of phenotypic mixing. The number of recombinants scored by this procedure was in good agreement with the number of Xc+ (speckled) plaques seen on mixed indicator. Since essentially all recombinational acts involving the minority parent will involve the majority parent as the other partner,

BY

LAMBDA

73

PHAGE

and so be revealed, the observed ratio of X+h/Xch in the progeny is multiplied by 0.5 to make the recombination data comparable with those obtained in crosses made under conventional conditions. RESULTS

Infection

under Anaerobic Conditions

Cooperative infection in the bulk of the K population, but not the acceptance of X. C by singly infected exceptional cells present in K populations, is dependent upon an aerobic environment during the initial stage of infection. This is illustrated by the results of the experiment described in Fig, 1, which compares the probability of successful infection of K cells by X. C as a function of the multiplicity of infection when the infection was carried out under aerobic and anaerobic conditions. The infections occurring at low multiplicities arise from exceptional cells, and their frequency is similar under both circumstances. Under anaerobic conditions, the infections occurring at high

‘0-4

I . 0.2

0.5

MULTIPLICITY

1.0

2.0

OF

5.0

I IO

INFECTION

FIG. 1. The effect of anaerobiosis on cooperative infection. Log-phase Escherichia coli K (W-3110) in ice-cold broth4.01 M MgSOc was distributed among a series of tubes to which Xch. C contained in ice-cold broth-MgS04 was added to bring the total volume to 1.0 ml. The contents of one set of tubes was then immediately gassed with Nz and the other with air for 5 minutes, while still ice cold. The tubes were then incubated for 20 minutes at 37” with continued gassing, iced, and successful infections determined. The cell concentration was 3.8 X lO*/ml. Phage adsorption was 98% complete.

74

WEINFELD

T~IE EFFECT

OF ANAEROBI~SIS

ON

AND PAIGEN TABLE I COOPERATIVE INFECTION

Experiment 1 Treatment .Aerated Open to air

Cells/ml during adsorption

PH~GEG

Experiment 2

Per cent of infected cells yielding phw

Cells/ml during adsorption

Per cent of infected cells yielding phase

2.40 x 108

2.24 2.20 0.45

2.26 x 108

1.4G 1.43 0.29

1.20 x 109

1.87 0.13 0.026

1.13 x

0.65 0.061 0.019

N2

Aerated Open to air N2

BY L.~MBD.~

109

a Two separate log-phase cultures of E. coli K (W-3350) were mixed ice-cold with x.C to give multiplicities of infection of 5.8 and 5.4, respectively. These mixtures were further diluted 1:5 with brothMgSOc . Aliquots of the original mixture and of the 1:5 dilution were aerated, allowed to stand open to the air, or gassed with Nt , respectively, at 37”, whereafter successfulinfections were determined. Phage adsorption was 9597Yo complete. The adsorption period was 10 minutes for the higher and 22 minutes for the lower ceI1 density.

nlultiplicities, which would predolninantly arise from cooperative infection, are nearly all prevented. Since cooperative infection is not recovered when the cells are returned to air under the usual conditions for plating infective centers, it is apparent that not only is there an energy-dependent step in cooperative infection, but that the X- C chromosome is irreversibly inhibited once exposed to the cytoplasm of a K cell under anaerobic conditions. The Absence of an Anuerob~ Erect on the The effect of anaerobiosis on the multiplication of x is specific for the condition of a host-modified phage entering a restricting cell. The subsequent replication of Xch.K and X.K in E. coli K and of kch.C and x 1C in E. coli C is unaffected by periods of anaerobiosis during and after phage adsorption, Formation of the infected complexes at multiplicities of infection from 4 to 9 under lYz, followed by incubation under Nz for periods up to 25 minutes before plating, resulted in plaque yields insignificantly different from those obtained when similar cells and phages were adsorbed and incubated under aerobic conditions prior to plating.

The Availubi~it~ of Oxygen during Adsorption When Standard Phage Techniques Are Used When placed in a Warburg apparatus in broth-O.01 M MgSOc at 37”, a culture of E. coli K (W-3350) exhibited a rate of oxygen consumption sufficient for lo9 cells/ml to exhaust the dissolved oxygen of the medium in less than 1 minute. Under such conditions the rate of diffusion of oxygen should become a limiting factor in the ability of X*C to exhibit cooperative infection. For this reason the infectivity of x .C in E. co& K was examined under the oxy~enation conditions that prevail when standard phage techniques are used; i.e., when no active aeration is performed and oxygenation occurs by passive diffusion. The results of two such experiments are presented in Table 1. It is apparent that at a multiplicity of 5 to 6, where cooperative effects predominate, the diffusion of oxygen becomes limiting only at higher cell densities. As would be expected at these higher cell densities, and under conditions of passive oxygen diffusion, the probability that a successful infection occurs becomes dependent upon the absolute volume of the adsorption mixture, and upon the shape and size of the

COOPERATIVE

TPJFECTION

eontainer in which adsorption occurs. At the cell densities (2 X ~Os/ml) and volume (0.5 ml) whieh were employed in the original demonstration of cooperative infection (Paigen and Weinfeld, 1963), oxygen diffusion was not a limiting factor. Th#eDevelopmentof Indi$erence to Anaerobic Block

BY LAMBDA

TABLE 2 EFFECTS OF Gas ALTERATIONS ON C~~PERAT~~E INFECTIOP

Treatment

Nz for 3 min. Ng for 20 min. When X. C infects a restricting cell under NZ for 3 min. then aeranaerobic conditions, the loss of the subated for 17 min. sequent ability to cooperate is irreversible. Air for 3 min. This fact makes possible an experimenta Air for 20 min. definition of one of the stagesin the process Air for 3 min. then NZ for 17min. of cooperation. This is the time when the in-

fectious cycle of X. C has proceeded to a sufficient extent that the infected complexes are no longer sensitive to anaerobiosis. As a necessary preliminary it was found (Table 2) that a period of exposure to nitrogen as short as 3 minutes at the time of infection is sufficient to prevent the subsequent multiplication of X-C. Similarly, a 3minute exposure to air during the time of phage adsorption is sufficient to protect against subsequent inhibition under anaerobic conditions. Reduction of the aerobic period to 2 minutes results in a slight loss in subsequent resistance to iYz. This estimate of the time required to deveIop indifference to anaerobiosis must be considered as lnaxilnal, since an appreeiab~e fraction of phage particles adsorb during the latter half of the adsorption period. It thus appears that, provided the cell is in an aerobic environment, the decision in favor of cooperative infection is made very quickly after the injection of X.C chromosomes into the K cell. The time required for this decision (2-3 minutes) is considerably shorter than the time which must elapse (10 minutes) before the X chromosome normally undergoes its first replication (S&haud, 1960). This result suggests that the development of indifference to anaerobiosis involves the direct conversion of the entering X. C chrolnoson~e to a new form. ~~~u~n~~l ~njec~~~nof E. co&iX by X-C It is known that the cooperative infection of K cells by X. C results in the production

7’n

PKAGE

Per cent of infected cells yielding phage Experiment Experiment 1 2 0.050 0.017 0.28

0.021 0.017 0.064

1.94 3.96 2.64

a.!23 4.83 3.02

a Log phase E. coti K (W-33~} in ice-cold preaerated broth~.~l k! MgSO* was distributed in 0.9 ml aliquots among 6 tubes. Three aliquots were gassed with Nz and 3 with air for 5 minutes while ice cold. With continued gassing, the tubes were transferred to a 37” bath, and after 1 minute X ch.C was added in a volume of 0.1 ml. After completion of the indicated treatments, the tubes were iced and successful infections were determined. Viable cells per tube were 1.21 X lo9 and 1.18 X log in experiments 1 and 2, respectively. Multiplicities of infection were 12.3 and 13.1, respectively. Over 99% of the input phage was adsorbed in 3 minutes. of h+K in a single cycle of growth with a

normal burst size (Paigen and Weinfeld, 1963) and, as has already been stated, the infection of a K cell by X. K is unaffected by anaerobiosis. The rapidity, therefore, with which a decision for cooperative infection is made raises the question of whether this initial reaction involves cooperation between the entering X. C chromosomes to form one or more X. K chromosomes by direct conversion prior to any replication. Alternatively, the first step might involvs the formation of a new chromoaomal species, X. C,, which persists through the anaerobic period, and which subsequently cooperates to permit replication and the production of X. K progeny. In atten~pting to distinguish whether the initial step represents the conversion of the x’C chromoson~eto X-K or to X-C,, experiments were performed in which the infecting phages were separated into two populations and introduced at separate times. If

76

WEINPELL)

AND

PAICEN

Per cent infected celIs yielding phage Expxpt.I Expt. XI

Condition x ----x Fro. 2. Sequential infection of E. coli K (W3359) by XchaC. The open bars signify an atmosphere of air, and the solid bar an atmosphere of Nf. R-l;, represent individual tubes, each containing 8.5 X 108 and 8.9 X 108 viable cells/O.9 ml in the two experiments, respectively, which were preaerated for 5 minutes while ice cold and then for 2.5 minutes a.t 37” prior to the start of the experiments, which continued at 37O. Phege added was contained in 0.1 ml sus~nsjou medium for singie aliquots and 62 ml for double aliquots. Suspension medium replaced the first pulse of phage in A and B. The placement of the phage symbol denotes the time of addition. A single symbol represents multiplicities of infection of 7.7 and 5.6 in the two experiments, respectively. After 20 minutes all tubes were iced, the contents were diluted with cold 0.01 M MgSO,, and aliquots were plated to score successful infections. Phage adsorptian was at least 980j0 complete in 3 minutes.

cooperation occurs during the first stage, to form with some finite probability a X+K ~hr~~~o~rn~~ then the introduction into a K cell of two populations of X - C chromosomes separated in time by a period of anaerobiosis should yield with equal probability twice as many x SK ehronloso~~~~. Since K chromosomes show no dependence upon cooperation, the overall probability of obtaining a successful infection in such a K cell would be twice that achieved with a single input of A. C. On the other hand, if the first stage represents the conversion of A. C to X. C, which persists through the amwobic period, the h.C, would still possess the ability to cooperate with the second input. fn this case the probability of producing a successful infection would be about 15-20 times that of a single input, which is the increase to be expected from doubling the number of phages available for cooperation (Fig. 1 >_

x

x

---x ----

h

0.3

1.0

5.9

9.0

0.4

0.9

0.6

1.4

a The experiments were performed essentially as depicted in Fig. 2. The duration of the first gassing period was 4 minutes, the second 7 minutes, and the third 16 minutes. The first phage aliquot was added at 0 minutes, and the second at 13 minutes. Under “condition” a thin line represents a period of active aeration of the culture, and a heavy line gassing under N?. M.o.i.‘s for a single phage aliquot and viable cells (W-3350) per tube were 2.4 phages per cell and 9.3 X 10s in experiment I and 3.0 and 8.6 X lo8 in experiment II.

E’igure 2 presents the results of two experiments in which the interval between successive infection of K cells by Xch *C was interrupted by a 7-minute period of anaerobiosis. Doubling the number of phages available for cooperation (3 vs. A> considerably increased the fraction of successfully infected cells. The interrupted addition of host-rnodi~~ phages produced no fewer infections (C> than did sinlu~taneous infection (B),2 and, most signi~~an~~ly, the ability of sequential infections to cooperate was not diminished by an intervening period of anaerobiosis (D). Similar results were obtained in five other experiments in which the intervening period of anaerobiosis was 2 As a matter of fact, the same number of phage always caused slightly more infections when they were introduced sequentially, rather than simultaneously. This probably arises from a eompetition between two reactions, the formation of X-C, and the tatal inactivation of X~C. The loss of X-C by ina~tivatiou is presumably lessened when the rate of phage entry does not exceed the capacity for hC, formation.

COOPERATIVE

INFECTION

BY LAMBDA

77

PHAGE

TABLE 4 COMPARISON OF X-C AND X+K AS SECONDARY INFECTING PHAGES=

XGh.C ------

Parameter Total number of successfully infected cells Percentage of successfully infected cells Fraction of successful infections yielding mixed bursts Phage yield (X10*) : A++ Xch x+h

Average burst size: Degree of phenotypie

x++.r

_-

1.5 x 10” 0.15

1.08

x

10.9

-

0.17

1.18

23 0.43 0.061

108

Xch-C -------

A++-K -

6.7 X lo8 68 0.10 430 0.94 1.6

xch

79

21 2.4

70 1.4

Xch x+h

-

3.3 10

4.7 115

A++ mixing:

Xch-C

a Log-phase cultures of E. e&i K12 ~-~~) were concentrated to 9.9 X lo8 cells in 0.5 ml of brothM,SOa. To this was added 0.1 ml Xch-C (m.o.i. = 3.1). The secondary phage was added 13 minutes later in a volume of 0.4 ml. (m.0.i. for X++.C = 16, X++.K = 4.4). Between minutes 4 and 11 the cultures were gassed with NZ , otherwise they were under active aeration. The experiment was stopped at 22 minutes by the addition of cold 0.01 M MgSO 4 , and cultures were subsequently diluted for direct pIating of infective centers and incubation to produce a lysate. Average burst sizes are calculated on the basis of the number of cells yielding that type of phage. The degree of phenotypic mixing is expressed as the ratio of titers on K12/X (CR63) with and without preadsorption to K12 (W-3350).

varied from 5 to 10 minutes, and the overall duration of the experiment from 10 to 20 minutes. In all cases the control infections showed that the anaerobic block was essentially complete within 3 minutes. It appears likely that the increase in the number of cells yielding progeny after sequential infection occurs becauseof cooperation between the two phage populations rather than from any loss of restricting ability in cells recovering from the primary infection. In separate experiments the number of successfulinfections was not increased above that produced by the first aerobic input of phage alone if the secondary infection occurred under nitrogen (Table 3). Thus, there was no increasein the number of special cells that are unable to apply restriction. Furthermore, as later experiments show, the phages entering during both the primary and secondary infection multiply together in the same cells. An appreciable percentage of successfully infected cells liberate progeny derived from both the pri-

mary and secondary infecting phages, and genetic recombination occurs between the two populations of phage chromosomes. The experiments on sequential infection suggest then, that the primary event in the infection of K cells by X. C is the formation of a new type of phage chromoson~e (X . C,). The new ~hromosomal species is distinguished from h ‘C by its stability to anaerobiosis, and from X. K by its subsequent requirement for a multiplicity dependent process of cooperation to permit successfulinfection. Secondary Infection by X-C and X+K The question whether the X4C chromosome is converted to h. C, has also been examined by comparing the processes of marker rescue and recombination when infection with X++ .C or X++ .K followed infection by Xch-C. If X+ f. C were converted directly to X++ .K it should produce a result qualitativeIy similar to that with X+ + PK. Table 4 shows the results of

78

WEINFELD

AND

PAIGEN

TABLE RECOMBINATION

AND

SC’PPRESSION

Ach + A++

Parameter

Total number of successfully cells Percentage of successfully cells Fraction of successful yielding mixed bursts Phage yield (X108): X++ Xch X+h Average

Apparent (%I

burst

sizes:

recombination

infected infected infections

X++ hch frequency

a Log-phase E. coli K12 (W-3350) infected with h++ C (m.o.i. = 6.4) indicated. The heavy bars indicate with that of Table 4. The apparent kfh relative to the minority parental

4.1

5

DURING

x

-

COOPERATIVE Xch

107

3.9

INFECTION

X++

1.7

x

Xch

10s

I

16.3

2.1

x

BY x.CP

-

x+t

A++

108

1.8

Xch

--

20.2

x

17.3

0.70

0.30

0.26

0.24

1.8 0.63 0.071

10.8 2.6 0.18

40 3.1 0.35

2.8 32 0.34

4.9 1.9

6.5 5.1

20 5.7

6.8 18

5.6

3.5

5.5

6.1

-

108

-

was concentrated to 1.04 X lo9 cells in 0.9 ml of broth-MgSO, and and kch. C (m.o.i. = 6.2) in volumes of 0.1 ml each in the sequences periods of anaerobiosis. In all other details the protocol is identical recombination frequency is calculated as one half the percentage of genotype in the yield, for the reasons discussed under Methods.

such a comparison. It can be concluded from these data that secondary infection by either X .K or X. C caused an increase in the number of cells yielding phages of the primary type, and a distinct reduction in the average burst size of the primary phage.3 Of particular significance for the question of X. C,, however, was the finding that the nature of genetic recombination between the primary and secondary phage was markedly different depending upon whether X ’ C or X. K was the secondary phage. When X+ + . C followed Xch . C, recombination between the c and h markers occurred with normal frequency. The 3 Because the average burst size of the primary phage is small relative to that of the secondary phage, estimates of the number of cells yielding mixed bursts are minimal. Some plaques had a morphology only slightly different from those plaques arising from one or the other of the two parental types. This effect probably arises from the unequal numbers of phage types released from the originating infected cells. Plaques which arise from bursts containing lower than average numbers of the minority type are probably scored as yielding only one phage type.

yield of recombinant X+h phages (0.061 X 10s) was considerably less than even the minority parent (0.43 X 108). In contrast to this, when X+ + . K was the secondary phage following AcheC, there were more recombinant type phages produced (1.6 x 108h+h) than there were nonrecombinant parental Xch type (0.94 X 10s). Moreover, the recombinant X+h progeny produced after secondary infection by X. K were considerably more subject to phenotypic mixing of the h character than were the nonrecombinant Xch produced under the same conditions. It appears, therefore, that the recombinants arising after X. K superinfection did not originate in the same cells that were multiplying Xch. These facts suggest that the dominant effect in secondary infection by X. K is the rescue of single markers at high frequency, whereas secondary infection by X. C permits the multiplication of the intact chromosome of the primary infecting phage. A similar effect of unmodified phage on the rescue of individual markers from modified phage was reported by Dussoix and Arber (1962) for the X.K-X.K(PI) host control system.

COOPERATIVE

INFECTION

Since the chro~nosonle form to which X-C gives rise behaves in a manner distinct from X- K during genetic recombination, these experiments support the conclusion that the 1. C, chromosome is an intermediate in the multiplication of a host-modified chromosome. Sequential Infection

BY

Secondary phageand

m.o.i.

ch ch ch ch ++ ch ch

6

EFFECT OF S~ONDA~Y INFECTION THI NUMBER OF CELLS YIELDING PRIMARY PHAGE~

Primary phageand

x x x X X x x

79

PHAGE TABLE

THE

by Xch. C and A+ + . C

Additional information on the interaction between host-nlodifi~ phage was obtained from experime~~ts on sequential infection in which primary and secondary infecting phage was distinguished genetically. Table 5 presents the results obtained when Xeh-C and X++ -C infected K cells either simultaneously or sequentially. It is apparent that during sequential infection, whichever phage comes last produces a distinct suppression of the first phage, and reduces its average burst size. This effect is somewhat more marked when X+ + *C follows Xch ’ C, since X+ + ’ C has some growth advantage even during simultaneous infection. (The growth advantage of X+ + 3C during “simulinfection probably reflects the taneous” fact that under such circumstances it enters the cell somewhat later on the average than does Xch. C. This is because h type phages adsorb more rapidly than h+ phages.) In control infections involving the sequential addition of unnlodified phages, it was always the primary infecting phage which had a growth advantage. When Xch-K and X+ + ’ K were added simultaneously, it was the Xch which predominated. Whether the entrance of the secondary phage will significantly increase the number of cells yielding the primary phage depends upon the multiplicity of infection of the primary phage, and the consequent fraction of cells which yield phage of the primary type in the absence of any secondary infection. Whenthe fraction of cellswhich would otherwise yield the primary phage was small, the addition of the secondary phage caused an increase in the number of such cells; when this number was large, the effect was slight. These data are summarized in Table 6.3 The total yield of progeny phage of the primary type is the resultant of these two

LAMBDA

(2.1) (3.1) (2.1) (2.4) (3.4) (6.9) (6.4)

Percentageof cells yielding primary phagewhen infected

m.0.i.

x h x A h x h

++ ++ ++ ++ ch 4-f ++

(9.2) (16) (3.1) (3.4) (2.4) (7.5) (5.8)

UPON THE

with

Primary alone

Primary and secondary

0.15 0.15 0.18 0.54 4.2 7.7 11.8

1.3 1.8 7.5 10.0 11.0 3.9 11.5

4 The data summarize the results of separate experiments. The experimental protocols were essentially identical with that described for Fig. 2. The adsorption mixtures contained 0.93-1.26 X lo9 cells in 0.9 ml + 0.1 ml for each phage suspension. The adsorption mixtures were actively gassed. The time interval between the primary and secondary infections was 13 minutes. In all but the last two experiments this included 7 minutes’ anaerobiosis starting 4 minutes after the primary phage was added. The total duration of the adsorption period was 22 minutes, at which time the mixtures were diluted and plated for infective centers on mixed indicator as described under Methods.

factors: the changes in average burst size and the number of yielding cells. The total yield of the primary phage could be either greater or smaller than that found when it entered alone. Under all circumstances recombination appeared to occur at approximately normal frequencies. Whether or not a period of anaerobiosis separated the entrance of the two phage types, and even when the primary phage entered under anaerobic conditions, apparent recombination frequencies were not affected. A summary of all the pertinent recombination data obtained under the various possible experimental configurations is given in Table 7. The results are self-consistent and seem to represent only a random scatter about a mean value of 4.8 ‘3. (These data were ob-

80

WEINFELD

RECOMBINATION

M.o.i. of X++: X ch: +

9.2 2.1

7 DURING

z

24 314

COOPERATIVE

138l

.;‘:4”

4.9

4.6

xch

x++

xch

A++

4.2 4.0

7.4

3.2

6.5

3.0

2:

kit

76::

3.8

4.8

5.5

4.9

5.6

xch

A++

INFECTION”

:.f

A++ xch

-

PAIGEN

TABLE OBSERVED

FREQUENCIES

i::

AND

3.5

7.1

3.0

6.6

5.5

2.9

0 A summary of the apparent recombination frequencies observed in various experimental configurations. The values are calculated as described under Methods and expressed in percentages. The infection sequence is indicated to the left, and the heavy bars signify periods of anaerobiosis. The experimental protocols were similar to those of Tables 4 and 5. TABLE RESCUE

OF ANAEROBIC

Total number of successfully infected cells Number of cells yielding Xch Percentage of successfully infectec cells Fraction of successful infections yielding mixed bursts Phage yield (X10*) : k+f kh x+h

Xch

X.

Ca

Xch --

A++

Xch

5.7 x 106

7.7 x 10’

7.5 x 107

5.7 x 106 0.06

1.0 x 10’ 7.9

7.5 x 107 7.7

-

0.13

0.34 -

19.5 0.60 0.059

60

Average burst size: X++ recombination

8 C BY AEROBIC

Xch I-

Parameter

Apparent (%I

X.

-

frequency

25 6 4.9

40

-

53 -

a Log phase E. coli (W-3350) was concentrated to 9.8 X lo* cells in 0.9 ml of broth-MgSO, and infected with kch.C (m.o.i. = 6.9) and h++.C (m.o.i. = 7.5) in volumes of 0.1 ml each in the sequences indicated. The heavy bars indicate periods of anaerobiosis. In all other details the protocol is identical with those of Tables 4 and 5. tained when simultaneous

Xch- C was followed

by or was

with X+ +. C. Although it is technically difficult to measure recombination frequencies when X+ + .C is the primary phage, and hence Xch is the predominant progeny type, this was done in several cases. Within the limits of experimental error, similar recombination frequencies were obtained.) Similar values for the recombination frequency between c and h

have been reported previously (Appleyard et al., 1956; Kaiser, 1962), and were obtained in this laboratory for control crosses of Xch.K by X++ .K. It should be remarked that when lysates from these crosseswere plated on a mixed indicator of sensitive K12 (strain CSOO) mixed with K12/X (strain CR 63) there were a variable number of “mottled” plaques obtained, in addition to the expected plaque

COOPERATIVE

INFECTION

BY

LAMBDA

PHAGE

81

The present results sharply delineate the two modes of successful infection by hostmodified X, and reinforce the earlier conclusion. Phage growth in special cells is not prevented by a period of anaerobiosis during and shortly after phage adsorption, whereas cooperative infection is strongly dependent on a supply of oxygen during the same period. The rapidity with which cooperative infection was irreversibly inhibited by anaerobiosis, and the rapidity with which the susceptibility to anaerobiosis disappeared when adsorption proceeded aerobically, strongly suggests that the X. C chromosome as such can survive only briefly once it enters a Rescue of X.C Entering Anaerobically restricting K cell. Its maximum survival time is estimated to be 3 minutes. Its fate Although X. C that enters under anaerobic under aerobic conditions appears to be conconditions makes no contribution as a version to a new form of chromosome, X-C,. secondary phage toward increasing the probThe existence of X. C, is suggested by the ability of successful infection, it can conenhanced cooperative infection resulting tribute genetically when entering as the from interrupted offerings of X. C separated primary phage. A typical experiment is by an anaerobic period, and by the genetishown in Table 8. It is apparent that a cally dissimilar consequences of secondary secondary infection by X+ +. C after Xch. C infection by X’K and X.C. The new species has entered under anaerobic conditions of chromosome differs from X. C in that its produced a large increase in the number of multiplication can no longer be blocked by cells yielding Xch, a decrease in average burst. anaerobiosis, and from X.K in that it is still size, and a normal rate of recombination dependent upon a cooperative process for between the two. These results suggest that growth. although phage chromosomes which enter During cooperative infection with an anaerobically cooperate poorly they are average multiplicity of only 2-3 phages of not completely lost to the system. They each type per cell, the same frequencies of can multiply in cells in which X. C, chromorecombination between the c and h loci were somes have overcome the barrier of host observed as at higher multiplicities. This restriction. implies that within any cell destined to yield progeny not only is the efficiency of DISCUSSION conversion of X. C to X +C, high, but also Because the plating efficiency of X. C on that essentially all the X. C, chromosomes K cells varied with the physiological state formed participate in replication and recomof the bacteria, Bertani and Weigle (1953) bination. At low average multiplicities the concluded that rare special cells in the popufailure of any appreciable fraction of the lation were capable of supporting phage input chromosomes to enter the mating growth when such cells were singly infected pool would cause the formation of signifiby X. C. Confirmation was provided by a cant numbers of progeny phage in cells in statistical analysis which ruled out the alterwhich only one of the parental types multinative possibility that such infections were plies, with a consequent decrease in the due to the existence of uncommon x.C apparent frequency of recombination. phages that could function in K cells withThe fate of the X. C chromosome entering out restriction (Paigen and Weinfeld, 1963). under anaerobic conditions is uncertain. At higher multiplicities of infection, phage Although the product formed has a greatly growth proceeds by a process of cooperation. reduced capacity for cooperation, consider-

types observed. Replating the phages present in such plaques produced an array of plaques showing various morphologies. In this sense the phages giving rise to mottled plaques behave like genetic heterozygotes. The number of mottled plaques observed was very variable. In some cases none were found; occasionally they were more frequent than the conventional recombinants. There was no obvious correlation between the frequency of their occurrence and any experimental parameter. At the present time the genetic structure and mode of origin of the phages producing mottled plaques is unknown.

82

WEINFELD

able replication occurs if it is followed by X. C entering under aerobic conditions. This behavior is probably accounted for by a partial yield of X. C, under anaerobic conditions. Low levels of cooperation are observed after anaerobic infection, and a reduction in the average number of chromosomes per cell should affect cooperative infection, which is very multiplicity dependent, much more than it should affect chromosome rescue. The loss in functional capacity which occurs under anaerobic conditions may proceed by way of the DNAdegrading system which restricting ceils can apply to host-modified phage (Dussoix and Arber, 1962; Arber et al., 1963). Although degradation of X. C DNA may be sufficient to account for host restriction under anaerobic conditions, the apparently quantitative formation of XSC, under aerobic conditions and its subsequent requirement for cooperation suggest that the primary host-restriction under aerobic conditions is in the functional limitation of X. C, chromosomes. It is this restriction that is overcome in cooperative infection, The surprising feature of the cooperative process that emerges from experiments on sequential infection is the suppression by later phage of those that had entered earlier. Suppression of this kind is the converse of the mutual exclusion observed in control expe~nlents when unmodi~ed phage infect sequentially. The mechanism of suppression is not clear at the present time. The recombination results obtained in the present experiments permit a choice between the various classesof models which can be offered as an explanation of cooperative infection. These are summarized in Fig. 3. In the first of these (threshold) the host cell contains some finite number of phage inactivat~g sites, either adsorption sites or phage inactivating enzymes, which are finally saturated at higher multiplicities. This permits the last phage entering to initiate phage multiplication. In this case all the progeny formed in any cell are derived from one, or very few, parents and apparent frequencies of recombination should approach zero. In the second class of models (dispersive) the entering phage are disrupted genetically and most of the seg-

AND

PAIGEN Threshold

expected recombination frequency

- 0%

Dispersive

-50%

Cooperative

“normal

FIG. 3. Possible models for successful infection by host-modified phage.

merits are barred from replication. Progeny are formed by assemblingparts derived from various parental chromosomes. This class encompasses models assuming either a physical disruption of the phage chromosomes,or merely regional blocks to replication with the formation of progeny by a copy-choice mechanism. In these eases, recombination frequencies would be expected to approaeh 50% for reasonably distant markers. In the last class of models (cooperative) all chromosomes survive, but local regions are randomly blocked in transcription. If a complete set of functional sites escapesrestriction, then phage multiplication is initiated and all chromosomes replicate. In this case,normal recombination frequencies would be expected. It is therefore the last model that is compatible with the evidence presented here. Previous evidence on the statistics of cooperative infection argued against the first model, but did not distinguish between the second and third (Paigen and Weinfeld, 1963). Since only lo-12 sites are estimated to be involved in cooperative infection (Paigen and Weinfeld, 1963), it would be most plausible to assumethat these are the sites needed to synthesize the minimal enzymatic apparatus required to initiate DNA replication. The function of these sites could be expressed only after the conversion to the X. C, chromosome. All other functions would be served by the unmodified progeny chromosomessubsequently formed. It is interesting to consider whether an “X” form occurs similarly as an obligatory intermediate in the replication of phage chromosomes in general, a form that is re-

COOPERATIVE

INFECTION

waled in the present case by virtue of the special circumstances of host-eontroll~ variation. ACKNOWLEDGMENTS The authors are indebted to Miss Patricia Lyons, Mr. Leonard Stauffer, and Mr. Francis Pacholec for excellent technical assistance. REFERENCES R. K., MCGREGOR, J. F., and BAIRD, K. M. (1956). Mutation to extended host range and the occurrence of phenotypic mixing in the temperate coliphage lambda. Virology 2, 565K?4 “, 1. ARBER, W., HATTMAN, S., and Dnssorx, D. (1963). On the host-controlled modification of bacteria. phage X. Virology 21, 30-35. BERTANI, G., and WEIGLE, J. J. (1953). HostAPPLEY.%RD,

BY LAMBDA

PHAGE

83

controlled variation in bacterial viruses. J. ~~c~er~oZ. 65, 113-121. DUSSOIX, D., and ARBER, W. (1962). Host specificity of DNA produced by Escheriehia coli. II. Cbntrol over kceptance of DNA from infecting phage X. J. Mol. Biol. 5, 37-49. KAISER, A. D. (1962). The production of phage chromosome fragments and their capacity for genetic transfer. J. Mol. Biol. 4, 275-287. PAIGEN, K., and WEINFELD, H. (1963). Cooperative infection by host-modified lambda phage. Viroloau

19. 565572.

SI!XHA&,- J. (1960). Developpement intracellulaire du coliphage lambda. Arch. Sci. (Geneva) 13,428-474. VISCONTI, N., and DELBR~~CK, M. (1953). The mechanism of genetic recombination in phage. Gelaetics

38,

5-33.

WEIOLE, J., MESELSON, M., and PAIQEN, K. (1959). Density alterations associated with transducing ability in the bacteriophage lambda. J. MOE. Biol. 1, 379-386.