Multiplication in Serratia of a bacteriophage originating from Escherichia coli: Lysogenization and host-controlled variation

VIROLOGY

32,

619-632 (1967)

Multiplication Escherichia

in Serratia coli:

of a Bacteriophage

Lysogenization

and

G. BERTANI, Department

of Microbial

Department

of Medical With

Host-Controlled

from

Variation’

B. TORHEIi\12

Genetics, Karolinska AND

Originating

Znstitutet,

Stockholm

80, Sweden

T. LAUREKT

Chemistry,

an appendix

University

of Uppsala,

Sweden

by G. BERTANI

Temperat,e bacteriophage P2, originally obtained from Escherichia coli strain Lisbonne, and usually grown on E. coli or Shigella strains, is also able to multiply in some Serratia strains. One of these has been shown to give stable lysogenic derivatives upon P2 infection. In passing from E. coli to Serratia and vice versa, PZ undergoes host-controlled variation. The base ratio in the DNA of the Serratia strain used is typical of Serratia strains (597, GC content,) whereas that, of I’2 DNA (50% GC content) is like that of E. coli DNA, even after the phage has been grown in the Serratia host. Serratia bacteria lysogenic for P2 do not, seem to contain numerous P2 DNA equivalents. The accidental formation of identical base pair sequences in DNA’s of similar or different base ratios is discltssed in the appendix. INTRODUCTION

We have accidentally observed that the temperate bacteriophage P2, originally isolated from a lysogenic strain of Escherichia coli, can multiply, however inefficiently, in a strain of Serratia, and have succeeded in isolating derivatives of Xerratia lysogenic for such a phage. Strains of Sewatia seem typically t#ohave different DNA base ratios from t#hose of Escherichia strains (see for example the tabulation by Hill, 1966). Chemical analy1 This work was aided by United States Public Health Service research grant AI-04390 from the National Institute of Allergy and Infectious Diseases, and by grants from t,he Swedish Medical and Natural Sciences Research Councils and the Swedish Cancer Society. The technical assistance of Mrs. L. Miirndal and Mr. H. Pertoft is gratefully acknowledged. 2 Holder of a fellowship from the Scientific Research Council of Norway during 1962. Present address: Norsk Hydros Institut,t for Kreftforskning, Det Norske R.adiamhospital, Oslo, Norway. 619

ses (L. E. Bertani, personal communication) had indicated for PW a DSA base ratio close to that of Escherichia. In view of the fact t’hat both the origin and the implications of t#he wide range of DNA base ratios among different bacterial species are still rather obscure (see discussion by Sueoka, 1964), we t’hought it worthwhile t’o explore the experimental potentialities of the Pd-Serratia system, particularly in regard to lysogeny. MATERIAL

AND

METHODS

The bacterial strains used and their symbols are listed in Table 1. Bacteria of strain Sa form very slimy, expanding colonies on the surface of agar. For single colony isolat’ion, in order to reduce the size of the colonies, plates with a very thin layer of TA agar, dried for a long time, were used preferentially. Alternatively, the plates were incubated for shorter times than the usual 16-20 hours, or incubated at lower temperatures. The temperate bact,eriophage P2 (Ber-

A20

BERTANI,

TORHEIM, TABLE

Collection number N-6 C-f Cla

C-14Y NCI’Y’ /

Sa-1

su-18 Sa-56 Sa-71 Sa-88 Sa-97

Sa-98 Sh-15 Sh-16

2665

I,AlIRENT

1

Origin and properties B strain C (Bertani and Weigle, 1953). (Good indicator for phage 1’2 A faster growing s&line of (T-1 9 streptom?-cirl-resistatnt derivative of (‘-1 (also carrying the fertility factor P originating in st raili K-f2) Obtained from (‘-la by lysogeuization with PZ c Slaph~lococct~ ujernlenlnns (:Jlicrococcus lysodeikfic~s), no. 2665, Xational Collection of Type Cldtrlres, London. Yellow pigmented, bllt cont,aining a minorit,y of violet pigmenird organisms, which were isolated and rlsed for DNA extraction. Also called Micrococc~t~ lrLle7r.s855 by RosypalovB, BohBEek, and Rosypal (1966). It,s CX content has been estimated from DNA heat-denaturation curves as 72.8% (Silvestri and unpublished, for Hill, 1965), 73.3y0 (Rosypalovh el al., 1966), and 72.9% (Bertani, the I)TA sample used here) A strain belonging to the collection of the late Mark H. Adams of New York University, and originally labeled by him “Serrafia marcetscenc strain R3 whit,e.” Although unpigmented, it cau give origin to pigmented variants. It is acGvely motile, prototrophic, and relatively iusensiiive to streptomycin (as compared, for example, with most Escherichia coli st,rains). Using high concentrations of strept,omycin (more thnll 500 pg/ml), it is possible to isolate from it fldlp resistant mrltants. It. produces a bacteriocill active against E. coli strain C. A doltblc mlltallt obtained in t.wo steps from Sa-2, It requires leucine and lysiue A mrdtiple mrltant obtained from Su-1. It, requires lerlcine (same marker as in Sa-f8) resistant and arginitre, and is st reptornycin Obtained from S-56 by lgsogenization with phage P2.C Obtailled from Sa-f by lysogenixatiou with PR or/ 1 c Sh Obtailrrd from S-88 ill the collrsc of a superinfection experiment (Table 5, esperiment A). It is Ilot lysogetlic, alit1 is sensitive to 1’2 Obtained from S-1, by lysogenizatiou with phage P2.C strain Sh (Bertani, 1951). The best indicator for phage P2 Shigella t/!/.senteriar A streptomycitr resistant derivative of Sh,-15 h’scherichia Escherichia

C-85

8iKD

co/i coli

strain

tani, 1951), its plaque type mutants i,fl, 1, and c (Bertani, 1954), and its virulent, mutants 1% eirl and 1’2 ~,:ir.~(L. E. Bert@ 1960) have been described before. After the present work was t,erminated, it was realized that previously unsuspected genetic changes had taken place in the series of wild-type P2 ly&es used in the course of this work. Two new mutational types (symbolized as lg and, for the double mut,ant, lg cc) had selectively accumulated in the lysates, pructicnll!, eliminat,ing the true mild type (Bertam, Choe, and Lindahl, manuscript, in preparation). Since t#hese mutants cannot, be distinguished from the wild t,>-pc on the ordinary I,B agar used for 1’2, the two mut,ations will be disregarded in what, folloms. One or bot,h, however, were present in all preparations 1abeIed as wild type in what

follows. The prophages in Sa-71 and Sa-98 certainly carry the Zg mutation. There is instead no reason to believe that t’he prcparations of the various other Pd mutank used were so affected. Wild-type P.2, I?2 ~1, P2 1, and P2 ul 1 also differ in their ability to form plaques on LB agar wit,hout added calcium, but wit,h various concentrations of sodium cit,rate (Bertani, Choe, and Lindahl? manuscript in preparation), f?!? rtl being the most resistant and I-‘,$ I and I’2 1.d 1 t,he most sensitive to high concentrations of citrate, and this propert(y was exploited where needed to confirm plaque t’ype classification. B:tct,eriophage plating for titration purpose was as a rule done on LB agar using a fully grown, aerat,ed culture of strain Cy-85 in nutrient broth ns the indicator, and

COLIPHAGE

MULTIPLICATION

standard phage techniques (Adams, 1959). For the classification of plaque types, however, strain Sh (fully grown, aerated cultures in tryptone broth) was used as the For optimal plaque type difindicator. ferentation the phage was adsorbed to the indicator bacteria for S-10 minutes at 37°C in the presence of 2.5 X 10e3 M CaClz before mixing with soft agar and plating. Furthermore a rather thick (and perfectly flat) layer of bottom agar was used. The host used as a rule in preparing phage P2 is E. coli C or some of its derivatives. In the text, whenever there can be doubt as to which host was used in producing a certain preparation of P2, the phage symbol will be followed by the host symbol, e.g., PZ.Sa for PW grown on strain Sa. Phage preparations were made either in LB with 2.5 X 10e3M CaClz or (particularly for wild type P2) in a glucose-mineral medium supplement,ed with casein hydrolyzate, and were later concentrat’ed and purified by differential centrifugation. Details of these procedures and also a concentration method based on precipitation of the phage by methanol, which was used in some cases, will be described in a future publication. A crit,ical detail must be mentioned here, however, i.e., the addition of potassium phosphate buffer (pH 6.8) to a final concentrat#ion of 0.2 M just before mass lysis begins, in order to reduce readsorption of the phage produced to cell debris or surviving cells. Difficulties were encountered in preparing good titer lysates of P2 in Sewatia (even when using PZ.Xa as inoculum), and severe activity losses were often not’iced in the course of the concentration and purification process. The cause of this was not investigated. For the various infection experiments cultures were grown in broth (LB, unless stated otherwise) with aeration to a titer of bet.ween 5 X 10’ and 10s/ml, concentrated by centrifugation, resuspending in the same medium with added 2.5 X 1(Y3 M CaClz (needed for P2 adsorption). Phage was added, and the mixture was diluted into broth with CaClz (and antiphage serum if necessary) 10-15 minutes later. Unadsorbed phage was usually measured by centrifuga-

IN

SERRAZ’IA

621

tion. Only techniques not given in Adams (1959) are described below. Colonies to be tested for lysogeny were picked and inoculat’ed individually in broth, in the 1 ml wells of special plastic containers fitting regular petri dishes (each container having 25 wells). These dishes were in cubated for a few hours and their contents later inoculated (using an inoculator carrying 25 stainless steel nails, fitting the wells of the plastic container) into regular agar plates already seeded with an appropriate indicator in t’he soft, agar. To eliminate the growth of the lysogenic bacteria which would obscure the plaques a streptomycin resistant indicat’or was used and a drop of a 5000 pg/ml solution of streptomycin was added to t’he soft agar (Bertani, 1951). When Xerratia was to be eliminated, twenty times higher concentrations of streptomycin were needed. With colonies of strains fully resistant to streptomycin, other drugs (sodium azide, chloramphenicol) and corresponding resistant mut,ants of the indicator bacteria were used with success. The rate of phage production by lysogenic strains and the burst size were measured in modified single burst experiments (Bertani, 1951), using eit’her the higher streptomycin concentration, or, less satisfactorily, sodium azide. The extraction of DNA from Serratia was carried out following the standard procedure described by Marmur (1961), without,, however, the isopropyl alcohol step. The complete procedure was used for the extraction of DNA from Micrococcus. Phage DNA was ext’racted from purified phage preparations by the phenol method of Gierer and Schramm following Mandell and Hershey (1960), and Thomas and Berns (1961). Preparative equilibrium sedimentation in CsCl density gradient was performed in a Spinco model L ultracentrifuge wit,h the SW39 rotor (36,000 rpm, 19 hours, probable rotor temperature: 20°C, ca. 250 pg DNA in 2.7 ml of CsCl solution per tube, for DNA; 25,000 rpm, 21 hours, probable rotor temperature: 2”C, 3 ml per tube, for whole phage) (Vinograd a,nd Hearst., 1962). Densities in t,he original solution and in some of

@2

BEI:TANI.

TORHEIiM,

the drop fractions collected were calculated according to the formula of Ifft, Voet., and Vinograd (1961) from refract,ive index I)~ values, determined with an Abbe refractometer. Equilibrium sediment,ation runs of DNA in C&l densit’y gradients were also performed in a Spinco model E Analytical Ultracentrifuge at 25°C and 44,770 rpm (Vinograd and Hearst, 1962). The runs were made in a 12-mm 4” sector cell of Kel F supplied with :L - 1” wedge window. The height of the liquid column during a run was 1.2 cm. The concentrat,ions of C&l were adjusted to homogeneous solution densities bet’ween 1.71 and 1.73, and t)he latter were then determined from refractive index measurements. The opt,ical densities of the homogeneous solutions at 260 mp due to DNA were between 0.3 and 0.6. Centrifugation times were kept bet,ween 24 and 2S hours. Recordings were made with st,andard ultraviolet optics. Films were exposed for varying t’imes and analyzed in a densitometer (Beckman Model RB Analytrol). Buoyant densities for different Dn’A prepa&ions were first det,ermined from the densit,y of the CsCl solut#ion before cerltrifugation and the banding positions using Eqs. (15) and (40a) given by Vinograd and Hearst (1962) (perrlpin Table 4). l&al cst,imates (prrl in Table 4) were obt,ained by subtracting the observed density differences from the density assumed for the reference DNA. DNA compositions are expressed in terms i.e.. t,he percentage of of &‘ 5% GC COrlteIlt”‘, all the DSA bases (in moles) represented 1~~ gunnine plus cytosine. Where it applies, the GC content has been calculated from the buoyant density of the DKA by means of :I well established empirical relat,ion (Schildkraut, Marmur, and Dots, 196%; Eq. 4). J/etlia userl. I,B brot’h and agar (Bertani, 1951), usually with Tryptone replaced 1)) K-Z-amine type B (Sheffield Chemical, Norwich, ;\‘ew York). Tryptone agar (TA) and trypt,one broth (TB) (Sasalti and Bertani, 1965). Purified phagc preparations were usually suspended in SSC (0.15 Jr KaCl. 0.015 JI sodium cit,rate pH 7).

AND LAUREST RESULTS

Multiplication

of Phaqe P2 in Serratia

Bact,eriophage 1’a.C (whether produced in the lgt’ic or in the lysogenic cycle of multiplication) gives few plaques (between 2 X lo-’ and 3 X lo-” as compared with the number of plaques obt,ainable on strain C) when plated on S’errafia strain Sa or one of its derivatives. Such plaques are much smaller than t’hose on Esche~ichia coli strain C, and oft’en poorly visible, which explains, in part), t#he great variabilit,y of the t#it,er as measured on Sa. The adsorpt#ion of P2 to Sa bacteria is reasonably good and could hardly be at’ t,he root of such a low efficiency of plating. In one-step growth experiments with P2.C infecting Sa bacteria, neither the latent period (30-40 minutes), nor the burst size (50-200) for those few bacteria (“yielders”) t’hat produce phage (as measured on t,he usual C indicator) are strikingly different from those determined with str$in C as the host. The number of such yielders can vary very much from experiment’ t’o experiment. Within an experiment it increases with t,he multiplicity of irlfect,ion~without,, however, showing any obvious saturation effect’ or cooperation between the infecting particles, up to multiplicit,ies of infection of 30 or 40 (Table 2). Even at t,he highest multiplicit,ies used, as many as 30-50 ‘5s of the bacteria survive the infection. At,tempts t,o demonstrat,c a genet#ic heterogeneity in t,he bacterial population wit,h respect’ to setlsitivity to phage PR.C failed. In a preliminary screening of a number of other ASel-ratia strains (kindly supplied by Professor R. W. Kaplan, of Frankfurt, and by Dr. W. II. Ewing, of At,lanta) several have been found on which Pg can form plaques. The hosts commonly used for phage I’R, E’. coli st,rain C, and Shiqella tlysenferiae strain S%, arc easily lvsogenized by it. With such hosts, most, oi the infect.ed cells lyse; of the cells that survive (of t,he order of 10 % w&h Pa wild type, the proport’iou being greatly affect#ed hy. conditiotw), the large majorit)!. give orlgln to c*olo~lies

COLIPHAGE

INFECTION

OF

PHACE

TABLE 2 Serratia WITH P2.C:

YIELDERS

AS

MULTIPLICITY

A FUNCTION OF

MULTIPLICATION

NUMBER OF

OF

THE

INFECTION”

MultiFraction of Effective” multiplici@ input Tube of in- yieldingbacteria phage plicity of Ratio n/m fection Infection Y n nz A n c 11

0.32 2.4 6.0 32.

2.0 1.9 3.7 1.5

X x x x

lo+ 10-z 10-Z 10-i

0.002 0.019 0.038 0.16

G.3 7.9 6.3 5.0

x x X x

10-a 10-z 1O-3 10-a

a An exponentially growing culture of strain Sa-f was chilled, centrifuged, and concentrated by resuspending in fresh broth. Aliquots of this suspension were mixed with phage Z’2.C and incubated for 10 minutes. At this t,ime a sample from each tube was diluted into broth containing phage-specific antiserum (K = l), and incubated for additional 10 minutes. Meanwhile, surviving bacteria and unadsorbed (nonsedimentable at low speed) phage were titrated. Finally, the serum tubes were titrated for phage yielders by plating with the usual C indicator. Parallel platings with Sa indicator gave essentially identical counts. b Input, bacteria (titer at 10 minutes in control tube) = 2.5 X 108/ml. Unadsorbed fraction of input phage = 3070. Surviving bacteria (colony formers) at the highest multiplicity of infection = 50%. Calculated according to the hypothesis that e-” = 1 - g.

a high proportion of lysogenic corltairlirrg cells. Attempts to lysogenize Serratia wit’h P2.C were at first, unsuccessful. Only when high multiplicit#ies of infection, and literally thousands of surviving colonies were tested, did it become possible to isolate some lysogenie strains. The majority of Sewatia cells surviving t)he infection give origin to colonies indistinguishable from uninfect’ed Serl-atia. The few colonies that on first isolation (e.g., from platings performed within an hour from t’he time of infection) produce phage, are not as a rule pure lysogenic clones, but contain a large majorit’y of nonlysogenic cells, indistinguishable from the uninfect,ed bact,eriu. Often, even when the infected cells had been kept in the presence of anti-P2 serum before plating and the solid medium was such as to minimize phage readsorpt,ion (no Ca added, high agar concentration,

IN SERRATIA

dry surface), colonies were found to produce fair amount,s of phage, and yet to contain no lysogenic cells, within the limits set by the number of daughter colonies that could be test,ed in practice. Only much more laborious experiments could tell whether these colonies are the result of secondary phage infection of originally sensitive colonies or reflect the exist,ence of unstable, self-reproducing prelysogenic states. Once isolated, Sorratia strains lysogenic for P2 appear to be as stable as any other P2 lysogen, even when kept in the presence of PW-specific antiserum for several transfers. Those tested in modified single-burst experiments (Sa-71 and S&98), produced phage spontaneously in bursts of normal size at rates of the same order of magnitude as those observed for similar lysogenic strains of Shi~ella or E. coli (Bertani, 1951, 1954; Six, 1959). Like other P2lysogens, lysogenic Sewatia strains still adsorb P2, are immune to it and t’o its weak-virulent mutant PZ virl, and sensit,ive to its strong-virulent mutant P2 v$. The efficiency of plating of P2 VI?, C OII lysogenic Serratia is not higher than 011 the original Serratia strain (i.e., the presence of the P2 prophage does not “help” the superinfecting phage). No gross compositional heterogeneity in respect to density in CsCl gradient equilibrium centrifugation was noted for the DNA of Sa lysogens (experiments in Table 4). Possible lysogenization by P2 of Serratia strains other than Sa or it’s derivatives has not been looked for. Host-Controlled Variation The phage produced by lysogenic Serratia does not seem to differ from P2.C in most respects (plaque morphology 011 indicators C and Sh, sensitivity to specific antiserum, sensitivity to heat,, buoyant density, dependence on calcium for plaque formation7 which is inhibited by the addition of sodium citrate). It does, however, plate on Serratia with higher efficiency than P2.C (lO-100% of the titer obtained on Escherichia coli C indicators). This property is not specially acquired through the lysogenization process: lysates of PW prepared in Serratia hosts show the same high efficiency of plat-

6’24

BERTANI,

TORHEIM,

ing. This property is lost upon passage of either phage on C hosts (Table 3). The phenomenon is t,herefore formally classifiable as a host-controlled modification of the adaptive type (Bertani and Weigle, 1953; recent review: Arber, 1965). It seems to be independent of t’he modification impart,ed to P2 by E. coli B (Bertani and Weigle, 1953) in that PZ.Sa plates 011 E. coli B, and P2.B on Sa, as P2.C would 011 either indicator. Lysogenization of Serratia by P2.Sa has not been studied quantitatively. What, has been done suggests, however, that the tendency-mentioned above-to give clones containing only a minority of lysogenic cells persists. The DNA of PW.Sadoes not seem to differ in buoyant density from that of P2.C (Table 4, experiments A and B), unlike t,he DNA of the respective hosts (see Discussion).

AND

LAURENT

Superinfection of Lysogenic Serratia Attempts to demonstrate genetic recombination by crossing appropriate mutant derivatives of our strain of Serratia failed, thus precluding the possibility of directly testing whether P2 prophage is attached to the bacterial chromosome also in lysogenic Serratia, as it is in R. coli. Attempt,s to demon&ate t’he presence of DNA with the density typical of P2 in t,he lysogenic Sevatia (Table 4, experiments C and D) also failed (110 evidence for a minor peak on t,he light’ side of t’he Serratia DNA band). One can expect t,hat’ these experiments could have easily detected t’he presence of P2 DSA if the latter represented 1 o/o of the original Sematia DNA. For DNA COIItents of 2 X lo-l4 g per log phase Serratia bacterium and of 3.6 X lo-l7 g per P2 particle (Mandel, 1967), the estimate above

TABLE

3

EFFECT OF PREVIOUS Hosrr B~WTERIUM (Serratia OR E. coli 6) ON THE HOST R.4NGE OF PHAGE PI

Phage preparationa

P2 P2 P2, P2, P2,

c, from C-147 rd 1 c, from Sa-88 from Sa-71 from Sa-98 plat,e lysate made on

Original efficiency of plating6

2.8 x (ca. 3.2 X 2.2 x 1.4 x

10-a 1) lo-’ 10-1 lo-’

.4fter multiplication” in E. coli strain C-la Factor of increaseC 5.6 7.5 7.8 1.2 1.0

X x x x x

lo4 105 lo” 105 105

Serratiad

P2, liquid lysate on Serraliae

made

4.0 x 10-l

1.8 x 104

Efficiency of platingb 1.0 x 10-s (10-Z to 10-a) 2.8 x lo-” 9.6 X lo-’ (2 x 10-Z to 6 X 1(Y4) (10-Z to 10-3)

After multiplicationc in Serratia strain Se-1 Factor of increasec 1.7 5.5 5.7 1.2 1.2

x x x x x

102 10’ 10” 104 109

2.4 x lo4

Efficiency of plating6 2.0 (5 1.4 2.6 2.0

x x x X x

10-l 10-1) lo-’ 10-l 10-1

3.5 x lo-’

a The preparations from lysogenic strains are supernatants obt,ained by centrifugation of a lysogenic culture in broth, followed by sterilization with chloroform. * Rat,io of average titer obtained with three Serratia strains as indicators (Sa-f, Sa-18, Sa-97) ijo titer obt.ained wit,h the standard indicator E. coli strain C-85. No obvious difference was observed between the three Serratia strains. When used as indicator, Shigella strain Sh-16 gave titers on the average greater t,han those on C-85 by a factor of about 1.3. Values in parentheses are based on low or inconsistent counts. c Cultures of either C-la or Sa-1 (10 ml broth wit,h 2.5 X 10e3 M CaClz in each flask on a shaker) were inoculated with phage preparations so as to obtain an input titer of about 200 phages per milliliter of culture, and incubated until lysis occurred or full turbidity was reached. The cultures were then ceutrifuged. The supernatants were sterilized with chloroform and assayed on C-85. The factor of increase is the ratio of the latter titer t,o the input titer. d Last, of a series of mass plate lysates made ou Serratia (Sa-I or Sa-18) starting originally from a single plaque of P2.C on Serratia indicator. e Lysate made in broth on Sa-1, using t.he supernatant of a culture of Sa-98 as t,he phage inoculum; later concentrated and purified by centrifllgation.

COLIPHAGE

MULTIPI,TCATIO?J TSBLE

EQUILIBHIUM

SEDIMESTATION

DNA

OF Serra!ia

IN

SEIZRAl’I.4

625

4 .IND

P2 DNA

IN

CsCl

DENSITY

GRADIENT

Bands observedb FXperiment

A

B

Content of sample (in addition to reference0 DNA)

P2.C DNA and Serrntia S-f) I)N4’

f;;’

13

Refelqfcea’

(3)-(l)

6.50 1.706 1.71G5

6.M 1.721

0.022

(3)-(z)

(strain

PZ.Su DNA

G.44

rc Pcm,,d Pde

1.699 1.7095

r

6.G8

G.86

Pen,>

1.735 1.7085

1.758

Prrl

C

Segtia

Lysogenic Serratia (strain Sa-98) DNA: a fract,ion (12% of band, -0.005 to -0.014 density units from t)he peak) obtained from a previous preparative equilibrium cent.rifugation in CsCl

r Perw Prel

6.Gl 1.719 1.7195

6.71 1.731

Lysogenic Serratia (strain S-98) DNA: a fraction (7.870 of band, -0.005 to -0.008 density units from t)he peak) obtained from a previous preparative equilibrium centrifugation in CsCl

r PC”lP

6.55 1.716 1.7175

-6.66 1.730

Prel

0.015 -

0.023 -

0.012

0.014

u From Micrococcvs strain NCTC 2665. b In no case was there evidence for gross heterogeneity of composition in the bands. c I)istance (cm) of band from center of rotor. d Buoynut density (g/ml) corresponding to band position, calculated from the density of the CsCl sample before centrifugation. e Buoyant density (g/ml) for material in band, relative to an assumed buoyant density of 1.7315 g/ml for t)he reference DNA [corresponding to 73.0% GC content according t,o the formula of Schildkrarlt, Marmlw, and I)oty (1962)l. ’ An ultraviolet absorption photograph of this sample at equilibrium is given by Laurent (1966).

would correspond to between 5 and 6 copies of PR DNA per bacterium. Another method t’hat might help deciding 011 the number of copies of PR genetic material present per ITsogenic bacterium consists of superinfectmg the lvsogenic cells wit,11 P2 genetically distinct &om the prophage t#ype and measuring the frequency of double lysogenizat’ion as compared tjo t’hnt of subst,itution (presumably by recombination) of prophage markers by t’he genetic markers of the superinfecting phage (Bertani, 1953, 1954, 19%; Bertani and Six, 1953). Using this met)hod, although several cases of substitution of prophage markers were observed, no stable doubly lysogenic clones were obtained (Table 5). In 15of t’he

9 colonies that produced t,wo kinds of phage at isolnt,ion (experiment,s A and B, Table 5), the phage of the superinfecting t’ype (turbid plaques, in these cases) was less than 10 76 of t’he to&l. In all cases where the analysis was carried far enough, streaking out such colonies and examining a large number of daughter colonies, it was possible to show that’ these colonies contjained a correspondingly small number of cells in which prophage markers had been replaced by markers of the superinfecting phage. For the one colony (d, experiment’ A, Table 5) which at isolation gave a higher frequency of phage of t,he superinfecting type, of 50 daughter colonies 15 produced only turbid plaques, 34 only clear plaques, and one again

626

BERTANI,

TORHII:I~I, TABLE

Strain Expeti ISuperin ment fected

,4Nl)

LAURENT

5

SGPERINFECTIOK OF LVSOGENIC Serratia. COLONY AN.\LYSIS~ g MultiI Exceptiona d Daughter ‘hage typl Superinfectcolonies colonies tested p’“:y carried ing phage and type of (phage spot 9 uperin observed 1 phage carried fectior

Genotypes of exceptional prophages observed

-_ A

Sa-88

P2 rd 1 c

ca. 6

P2.C

1825

1 pure c+ 4 mixed c and c+

1 without phase B

C

Sa-88

Sa-98

P2 rd 1 c

P2

P2.C

P2 rd 1 c.C

200

cu. 30

ca. 7

cit. 8

2000

894

50, all c+ a) 1 c+, 49 c b) 50, all c c) 2 c+, 45 c d) 1 mixed, 49 c (see text) e) 1 mixed, 49 c (see text) 50, all c

Id rtl rtl l-d rd

1 1 1 1 1

Prophage loss

I-

1 pure c+ 5 mixed c and cc

1 doubtful (mixed?)

1’2 P2 P2 P2 P2

25, all c+ a) 2 c+, 123 c b) 1 c+, 74 c c) 2 c+, 73 c d) 15 c+, 34 c, 1 mixed (set text.) Sensitive to P: s

P2 rtl 1 P2 i-d 1 P2 Id 1 not analyzed not analyzed

P2 rd 1 c

/

i 0 A culture of lysogenic Serratia (in experiment, C started using a single colony as inoculum) in trypt,one broth with 2.5 X 1CF M CaClz in the log phase was concentrated by centrifugation and mixed with phage. After 20-40 minutes, the amount, of unadsorbed phage was measured. Anti-P2 serum was later added. The infected cultures were then diluted and kept growing for several generations (sixth column in Table), after which they were plated for colonies on agar. After incubation, colonies were picked and tested (see under Material and Methods) for phage t,ype carried, the differentiation at this stage fluctuations occur from experiment being primarily between c, c+, and mixed phage spots. Although to experiment and syst,ematic count,s were not made routinely, the average number of plaques per spot can be taken to have been between 15 and 20. Colonies differing from the original t,ype in respect lo phage produced were analyzed further to identify the plaque type. In some cases they were streaked out on agar and a sample of daughter colonies was analyzed in the same manner for phage produced. In experiment B 1990 colonies from the uninfected culture were also picked and tested in the same manner. All gave c spots as expected. a mixture. Upon further analysis of this clone (and of two similar cases in experiment B, Table 5), however, we did not succeed in isolating doubly lysogenic clones. It cannot be excluded that such rare subclones giving both kinds of phage be due to technical errors in the inoculation or handling of the multiple containers. These results are compatible with the existence in lysogenic

Sevatia of one prophage per bacterial nucleus (and no easily occupable secondary prophage site). Alternatively, doubly lysogenie bacteria, if they occur at all in this system, could be much less stable than those obtainable with the same phage from EJ. coli or Shigella. Since the data obtained in these experimenk, when compared with similar data

COLIPHAGE TABLE

I.

6

SUPERINFECTION OF LYSOGENIC PLAQUE ANALYSIS”

Experiment Strain superinfected Phage type carried Superinfecting phase Input, bacteria Input phage ~_____

MULTIPLICATION

Serratia. B

A

P2 rd 1 c P2.Sa

Sa-98 PZ P2 rd 1 c.C

105/ml 2.3 X lO*/ml

9 X 105/ml 3.8 X lO*/ml

Sa-88

.-

First supernatant Total plaques scored I Not scored of which

18,000 3 clear

Second supernatant Total plaques scored of which

12,ooo 29 turbid

15,000 0 clear

Third supernatant Total plaques scored of which

9,440 15 turbid

44,ooa 3 clear

19 rd I+ g+++ 1+1+

Genotypes of plaques analyzed and their occurrences

5ttc lrdlc

L

a A culture of lysogenic Serratia (started using a single colony as inoculum) in broth with 5 X lO+ M CaC12 in the log phase was diluted to about lo5 bacteria/ml in t,he same medium to which superinfecting phage in large excess was added, and incubated till fully grown. (This method does not permit a reliable estimate of the multiplicity of superinfection. It was used to obviate t,he preparation of P2 lysates with high titers, which is difficult in Serratia and with the rd I markers). The fully grown culture was treated with anti-P2 serum (to inactivate all free phage that might be present) and stored overnight in refrigerator. Next day the culture was diluted 1:2OO in broth, and incubated on a shaker until fully grown. It was t,hen diluted I:40 in broth and incubated on a shaker for 2:30-3:00 hours. At this time a sample was taken and centrifuged, the supernatant being sterilized with chloroform and stored. Another sample was diluted 1:40, and the procedure was repeated twice. Large volumes (200 ml each) were used for these four serial cultures (the 1:200, 1:40, 1:40, 1:40 dilutions) to reduce the effect of statistical fluctuations in sampling. The three supernatant,s obtained were plated on Shigella indicator, with preadsorption. The c and cf alleles can be easily scored even on crowded plates. Plaques

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SERRATIA

627

previously obtained with E. coli and Shigella, suggested a discrepancy with respect to the recombination frequencies for the markers used, a less laborious technique (described by Six, 1961) was applied to clarify this point (Table 6). The recombination data obtained in the two sets of experiments are summarized in Table 7, and compared with a summary of recombination data extracted from previous work. It is evident that for the same set of phage markers the recombination frequencies obtained in Serratia differ from those obtained in E. coli or Shigella. DISCUSSION

We have shown that P2, a phage which originated from Escherichia coli, can multiply, and establish a stable lysogenic condition, in Serratia. Wit’hout satisfactory methods for studying recombination in Sewatia, it was impossible to demonstrate the presence of a typical (i.e., attached at a specific site on the host chromosome) prophage in the lyeogenic Serratia strains obtained. The more indirect evidence [(l) stability of such strains; (2) recovery in superinfection experiments of clones lysogenic for a recombinant prophage, but not of doubly lysogenic clones; (3) failure to detect DXA with the density t,ypical of P2 in the lysogenic bacteria] is, however, consistent with such an interpretat,ion, and does not support the possibility’ of a nonchromosomal, multiple-copy st,ate for the prophage. The interesting point in this cormection is the difference in DNA composition between, 011 the one hand, Serratia strains [53.559.0 %, average 57.4 %, GC content; see Colwell and Mandel (1965) and Hill (1966)], specifically strain Sa (59.0% GC content, based on the reference DNA used; see Table 4) and, on the ot’her hand, P2 (50.0% GC content; from Table 4) and its better hosts, E’scherichia coli strain C [49.80/o GC content (Eigner, Stouthamer, van der Sluys, and Cohen, 1963), and Shigella dysenteriae of the minority type were picked, reisolated once, and scored for the other markers present. These minority type plaques are assumed to result from prophage or prophage marker substitution.

6z3

BlCRTANI,

AND

LAURENT

Incidence of prophage -~~ “Prophage substitution” types (222)

Keference

Host bacterium

Shigella

TORHEIM,

Bert ani (1954), Table

1, esp. b

18

Other trpes

1 (221) 1 (112) 1 (112) 1 (112) 3 (201)” 2 (102)" .~-~~ .~~~ 0 w 0

Bertani Bertani

exp. d (1956), Table 2 and Six (1958), Table 6, exp. a

E. coli c

Bertani

and Six (19581, Table

Serrafia

This paper,

Table

5, exp. A exp. B exp. C

0 0 1

5 (112) 3 (112) 0

This paper,

Table

ti, exp. ki

8

19 (112) 1 (212) 5 (112)

exp. Bd

6, exp. b exp. c exp. e

8 22 17”

exceptional tl-pes __

__10 8c 5

1

-

a With some exceptions noted below, the markers employed were rd, I, e and their wild-lype alleles. As they appear in different combinations in different experiments, to simplify comparison, symbol 1 stands for the allele present in the prophage state, symbol 2 for the allele present in the superinfecting phage. The symbols are written in parent,hesis; t)he order is always rd I c. Occurrences of double lysogenization (in Shigelba and in E. coli C) are not included in the summary. The results of superinfection of doubly lysogenic strains are included; in such a case, however, recombinations or substitutions at prophage locations different from the preferred one are not, included. Recombinant or substitut,ed prophages obtained in the course of the analysis of “unsegregat’ed” (mixed) clones are included. The dat,a for Shigella and E. coli are all from analyses of individual colonies. Ample data from plaque scoring (same method used in the experiments of Table G in this paper) exist also for such strains (Six, 1961), and confirm the results obtained from colony analysis. Note that in the experiments with E. coli and Shigella, as opposed to those with Serralia, no select,ion for substitut.ion of the c gene was applied in scoring for exceptional types. In comparing the t,hree sets of data, one should therefore disregard those prophage types whose sigla ends in 1. fJThe I marker was not used here (at least not, in the preferred site prophage, nor in the superinfecting phage). This is indicated by the symbol 0. Since rd and I appear to be rat,her closely linked as compared to c, it is probably safe to include these data in the summary in this way. c Instead of I, the marker b u-as used in this experiment. Marker b also appears to be closely linked to rd (Bert&, unpublished) and resembles I in phenotype. Hence the inclusion of these data. d These data are obtained with t,he plaque-scoring method, and thus are not strictly comparable to the rest of the table. Nevertheless they confirm the trend.

lysogenization occurs only when phage and S-15 [SO%:’ GC corlterlt (Rlarnlur and Duty, 1962; their “strain 15”) or, in host DKS’s have similar GC contents. The general, Eschekhia strains [49.8-53.6 OJC ; see number of phage-host systems on which Belozersky and Spirin (1960) and Hill (1966)] such a statement can be based is, however, very small. For one such system, Hershey and Shigella strains [49-53.5 71; see Hill (1963) has demonstrnt,ed segmental base (1966)]. It has been proposed-for example, by ratio heterogeneity in the phage chromosome, and pointed out the appurent~ly XLarmi (1960) and by I,uria (1962)--that,

strain

COLIPHAGE

MULTIPLICATION

cidental nature of the observed overall base ratio similarity between phage and host bacterium. Although similarity of base ratio for a t.emperate phage and its host might be a likely result of evolution in a lysogenic system of old standing, it is not necessarily a requirement for lysogenization in Campbell’s (1962) model of episome integration via circularization of the episome and reciprocal crossing-over between the latter and t’he host chromosome. This model requires homology (i.e., identity or similarity of base sequence and hence of base composition) only between a short segment of the episome (episit’e) and a corresponding segment of the host chromosome (chromosit,e). The simplest form of the model would also predict that different segments of a phage chromosome may be used as episites in different hosts or at different at’tachment point’s within the same host. Information on the length of such sites is very scanty. For phage X, it, has been estimated from genetic experiments to be about We0 of t,he total length of t’he phage chromosome, i.e., about 406 base pairs (Calef, Marchelli, and Guerrini, 1965). Since some episomes are known to be able to incorporate fairly long segment’s of the host chromosome thus becoming homologous to it over long base sequences, what one really would like to know is the minimum site length that still permit’s integration via recombination to occur at a detectable frequency. Very general arguments, unfortunately still of doubtful validity (Appendix to this paper; Thomas, 1966), suggest that such sites need not be very long, so that accidental homology permitting recombination between an episome and the chromosome of an “unnatural” host would not be hopelessly infrequent (see Appendix). The extent of annealing of heat-denaturated nucleic acids to form hybrid doublestranded structures (Schildkraut, Wierzchowski, Marmur, Green, and Doty, 1962) has recently been used by several workers as an estimate of the amount of common sequences in DNA’s of different origins. It has been found, for example (McCarthy and Bolton, 1963), that about 7% of the

IN

SERRATIA

629

E. coli DNA could react in this way with the DEA of a Serratia strain. An application of such techniques to the system described here ought to be very rewarding. In particular it might show more convincingly the presence of only one copy of the prophage per host genome. The unexpected results of the superinfection experiments, suggesting different linkage relationships for the same set of phage markers in lysogenic Serratia as opposed to E. coli or Shigella, deserve to be examined further. At this stage they can still be interpreted in different ways: either (a) the superinfecting phage DNA is fragmented by endonucleases more often in Xerratia than in the other two hosts, before it can pair and recombine with the prophage, or (b) pairing between superinfecting phage DNA and prophage DNA is irregular and is affected in different ways for physiological reasons in the two hosts, or, most interesting, (c) the prophage in Serratia is attached via a different episite from that used in E. coli. In this case the prophage map would differ in the two hosts and might lead to different linkage relationships in recombination with a superinfecting phage. If (a) were true, one might expect the effect to disappear when superinfecting phage grown in Sevatia (i.e., “adapted” to Serratia) is used. Instead (Table 7) the effect did not disappear. One can still assume, however, that the protection from degradation afforded the phage by the “adaptation” (hostcontrolled modification) is not in t)his case complete. Finally, one should perhaps point out an inherent practical limitation of the P,%Serratia system: the plaques of Pd on Serratia are very small and poorly visible, probably as a consequence of the luxuriant growth of the bacterium and its motility. In continuing work, it would seem advisable first to Dry to isolate nonmotile, rough mutants of Serratia, in the hope that they might give better plaques. APPENDIX

By G. BERTANI

In connection with “unequal crossing over” and more specifically with the attach-

630

BEXTANI,

TOI:HEIM.

ment of episomes to chromosomes according t,o Campbell’s model, it is interesting to consider estimates of the probabilities of occurrence of specified base pair sequences in DNA-as a prerequisite for pairing-and how they might be affected by different DNA compositions. The simplest approach is to assume (a) no constraints in t’he sequence, and (b) no phylogenetic relationships between the recombining partners. Hypothesis (a) is certainly incorrect [Josse, Kaiser, and Kornberg (1961), J. Bid. Chem. 236, S64)]. The various deviations from randomness observed to date seem however to be small. In first approximation, t’hey shall be neglected here. Hypothesis (b) is obviously unjustified. It should be point,ed out however that if significant deviations from the predictions t,hat, one might obtain by means of such an approach were in fact observed, they would represent objective evidence for common evolutionary origin or for convergent evolutionary development. In what follows, DNA base pair sequences will be considered to be generated by successive random extractions from a universe of base pairs having a given GC content, with t’he two possible orientat8ions in respect to t,he sequence being generated (i.e., GC versus CG) having equal probabilities of occurrence. Assuming for the moment that t’wo identical subsequences (‘$ites”) of length 1’ base pairs are all is needed for recombination to take place bet’ween two sequences (the host chromosome and the episome) of lengths H and E base pairs, respectively, one can calculate how frequently pairs of identical subsequences should occur when t’he sequences are generated by the mechanism assumed above. Imagine a specified long base pair “test sequence” of equimolar base composition. Imagine generating a second sequence by t,he method described above. After each extraction, the base pair obtained is to be compared with the corresponding base pair in the test sequence. The probability 11that t,hey are identical (“matching”) is obviously ;i. iZ~l uninterrupted succession of 1’matchings corresponds to the presence of two identical sites of length 1’ in t’he two sequences, at, corresponding posit,ions. I’or

,4NI) LA1JI:ENT

long sequences, such an event will repeatedly occur with a probability of u = [(I - p)p’]/ (1 - p’) per ext’raction [see Feller, W. (1957). “An Introduction to Probability Theory and Its Applications,” 2nd ed., Vol. I, pp. 279 and 299-301. Wiley, New York. Theory of success runs]. We are interested, however, in all possible sites common to the two sequences, whether they are at corresponding positions in the sequences or not. If fl is the length of the test sequence, and the latttter is imagined as a circle, there will be E different ways of comparing the sequence being generated with the test sequence. The average number of occurrences of pairs of identical sit’es will then be uE. If the sequence being generat’ed is longer than E’, the process will be automatically continued after the Eth extraction. If the sequence generated has length H, one can expect a total of uEH sites of length r ident’ically present in the new sequence and in the (circularized) test sequence. Further, since the direction of the two sequences is probably immaterial to t’he effect of pairing, one should take into account also all possible comparisons with the test sequence taken with t’he opposite direction. The total number of identical sites to be expect,ed will thus be 2 uEH. One does not know what is the length 1 of the shortest, site permitting recombination between episome and host chromosome to occur with a practically detectable frequency. On t,he basis of t,he above considerations, a guess can be made as follows. Of t,he episomes studied to date in some detail in respect to specificity of attachment sit,e, phages X and @SOare each known to attach as prophages at one chromosomal site only, specific for each of t,hem; phage P2 can attach at anyone of at least half-adozen sites, but shows definite preference for certain sites; the sex factor F can become integrated in the chromosome at many different sites in the host chromosome. Although the available evidence is far from satisfactory, t#hcdat,a available suggest [RIatney, T. S., Goldschmidt, E. I?., Erwin, S. S., and Scroggs, R. K. (1964), Biochewl. Biophys. Res. Co~n~wun. 17, 27S] that the passible integration sites for F might be limitctl in number to perhaps :50. 01~1 would say,

COLIPHAGE

MULTIPLICATION

from the scant’y information existing to date, that an E. coli episome has in common with the host between 1 (by definition not less than 1) and 30 site sequences. Assuming for a generalized episome E = 5 X lo4 base pairs, and for the E. coli chromosome H = 5 X lo6 base pairs, one finds t’hat values of 2 uEH bet#ween 1 and 30 can be obtained for values of 1’ bet,meen 17 and 19. Alternatively, one can imagine that a given episome, perhaps owing to the requirement,s of the recombination process involved, has only one site of length I which can be used for attachment to identical chromosites. In this case (i.e., 2 uH between 1 and 30) the corresponding 1 values are found to be between 9 and 11. Given a specified sequence of length 4r with equal representation of the four base pairs, the probability PE of constructing an identical sequence with 4 Y extractions from an E. coli base pair universe is obviously (JLJ~~. The probability PS of constructing the same sequence wit’h 4 1’ extract,ions from a Xewatia base pair universe (assume 60% GC content), is:

The ratio Ps/PE can be taken as a measure of the relative difficulty of obtaining E. coli sequences in Serratia by such a process. One finds, for example, I’s/PB = 0.66 for 4 r = 20. The ratio is st’ill greater than 0.1 for 4 1’ = 100. One can imagine “making” Xerratia out of E. coli by changing into GC (or CG) one-fifth of all AT (or TA) base pairs, chosen at random. The probability that a “typical” (i.e., containing exact,ly 5 r GC, CG and 5 1’ AT, TA base pairs) E. coli sequence of length 10 Y remains unchanged in this process would be ($<)5p, i.e., about 0.1 for 10 1’ = 20. REFERENCES ADAMS, M. H. (1959). “Bacteriophages.” Interscience, New York. ARBER, W. (1965). Host-controlled modification of bacteriophage. Ann. Rev. Microbial. 19, 3G53i8. BELOZERSKY, A. N., and SPMN, $. S. (1960). Chemistry of t,he nucleic acids of microorgan-

IN

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631

isms. In “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 3, pp. l-17-185. Academic Press, New York. BERTANI, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenir Escherichia co/i. J. Bacterial. 62, 293-300. BERTANI, G. (1953). Lysogenic versus lytic cycle of phage multiplication. CoZd Spring Harbor sgnp. Quark Biol. 18, 65-70. BERTANI, G. (1954). Studies on lysogenesis. III. Superinfection of lysogenic Shigella clysenteriae with temperate mutants of the carried phage. J. BacteTiol. 67, 696-707. BERTANI, C:, (1956). The role of phage in bacterial genetics. Brookhaven Symp. Biol. 8 (“Mutantioii”), 50-57. BEK~AXI, G., and SIX, E. (1958). Inheritance of prophage P2 in bacterial crosses. Vi’irologg (,, 357381. BEIIT.~~-I, G., and WEIGLE, J. J. (1953). Host-controlled variation in bacterial viruses. J. Bacteriol. 65, 113-121. BERTANI, L. E. (1960). Host-dependent induction of phage mutants and lysogenization. F’irology 12, 553-5G9. CALEF, E., MARCHELLI, C., and GUERRINI, F. (1965). The formation of superinfection-dollble lysogens of phage A in Escherichia coli Kl2. t’irology 27, l-10. CAMPBELL, A. M. (19G2). Episomes. Atlc. Genet. 11, 101. COLIVELL, R. IL, and MANDEL, M. (1965). Adansonian analysis and deoxyribonucleic acid base composition of Serratia marce.scens. J. Bacterial. 89, 454-461. IX~cma, J., STOUTHAMER, A. H., VAN UER SLUYS, I., and COHEN, J. A. (1963). A study of the 70s component of bacteriophage +X174. J. Mol. Hiol. 6, 61-8-l. HERSHEY, A. L>. (19G3). Annual report of the director, Genetics Research Unit. Carnegie Inst. Washington Year Book 62, p. 484. HILL, L. It. (19GG). An index to deoxyribonucleic acid base compositions of bacteria species, J. Gen. Microbial. 44, 419-437. IFFT, J. B., POET, I>. H., and VINOGILID, J. (1961). The determinat,ion of density distributions and density gradients in binary solutions at eqrlilibrium in the ultracentrifuge. J. Ph!/s. Chem. 65, 1138. LANXI, F. (1960). Genetic significance of microbial DN.4 composition. Perspectives Biol. Ued. 3, 418-432. LAURENT, T. (19GG). Bestiimning av molekylvikter. In “Experimentell biokemi” (by K.-B. Allgllstinsson). Svenska Bokfiirlaget, Stockholm. LUMP. S. E. (19G2). Molcc~ktr aui gcuetic

632

BERTANT.

TORHEJM,

criteria in bacterial classification. Kecenl Progr. Microbial. 8, 604-616. (University of Toronto Press.) MANIEL, RI. (1967). Infectivity of phage P2 DNA in presence of helper phage. lkfolec. Gen. Genelies 99,88-96. MAXDELL, J. D., and HERSHEY, A. 1). (1960). A fractionating column for analysis of nucleic acids. An&. Biochem. 1, GG-77. MARMUR, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. BioZ. 3, 208-218. M~I~M~R, J., and DOTY, P. (19G2). Determination of the base composit,ion of deoxyribonucleic acid from its thermal denaturation temperature. J. ~VoZ. Biol., 5, 109118. MCCARTHY, B. J., and BOLTON, E. T. (1963). An approach to t,he measurement of genetic relatedness among organisms. Proc. M&Z. ii&. Sri. U.S. 50, 156-164. I
AND

LAlJRENT

SCHILDKRAUT, C. L., WIERZCHO~SKI, K. L., MARMUR, J., GREEN, D.M.,and DOTY, P.(1902). A study of the base sequence homology among the T series of bacteriophages. ViroZog?/ 18, 43-55. SILVESTRI, L. G., and HILL, L. 1:. 1965. Agreement between deoxyribonucleic acid base composition and taxometric classification of Gram-positive cocci. J. Bacterial. 90, 136-l-10. SIS, E. (1959). The rate of spontaneous lysis of lysogenic bact,eria. Virology 7, 328-346. SIS, E. (1961). Inheritance of prophage P2 in superinfection experiments. ViroZogZj 14, 220233. SUEOKA, N. (1964). Compositional variation and heterogeneity of nucleic acids and protein in bacteria. In “The Bacteria” (I. C. Gunsaliis and 1~. Y. Stainer, eds.), Vol. 5, p, 419. Arademic Press, New York. THOMAS, C. A., Jn. (1966). Recombination of DNA molecules. Progr. h’ucleic Acid Res. Mol. BioZ. 5, 315-337. THOMAS, C. A., Ju. and BERNS, K. I. (1961). The physical characterizat,ion of DNA molecules released from T2 and T-l bacteriophage. J. !lfoZ Biol. 3, 277-288. VINOGRAD, J., and HEARST, J. E. (1962). Equi librium sedimentat.ion of macromolecules and viruses in a density gradient. For-l&r. Chem. Org. Naturstoffe 20, 372-422.