STUDIES ON THE MECHANISM OF TRANSDUCTION BY +y. I. GENETIC CHARACTERIZATION OF THE TRANSDUCING SEGMENT

JEAN-PIERRE GRATIA

Laboratory of Microbiology, Faculty of Medicine, University of Brussels] Manuscript received April 30, 1976 Revised copy received August 26, 1976

ABSTRACT

The bacteriophage +y, though related to the lambdoid phage +SO, has unusual features in its specialized transduction and is being investigated to determine the mechanism of the transduction process. Genetic analysis of the transducing element gives evidence for a relatively long and uniform linear segment, up to about 1% of the E. coli chromosome, extending in either direc- tion from the attachment site, e.g., on the right side: att80-to&- trpABCDE-cysB-pryF. The att end includes a variable amount of phage , probably very short in most particles. In a small fraction of the transducing particles the phage segment may be more extensive and, con- versely, the bacterial segment is shorter, ending around cysB. The transducing segment from modificationless carries a site susceptible to the K-restric- tion system which affects the efficiency of transduction.

EMPERATE bacteriophage $7 is capable of a high rate of transduction spe- Tcific for genes of the host adjacent to the phage attachment site. The trans- duced genes enter the recipient chromosome by a substitution recombination event, not by the addition of a phage genome to which host genes have been fused. It is thus the generation of the transducing particles which is extraordi- nary. These arise either following induction of the +y prophage or during lytic infection by +y. Transitory lysogenization during lytic infection, involving re- pression and then derepression, can be ruled out. Nevertheless, the transducing particles are only formed if the infected bacterium has the chromosomal attach- ment (att) site for the phage and if the infecting phage is Int+ (GRATIA1973). As shown below, the transduced genes include a relatively long linear se- quence, up to 1% of the E. coli genome, extending in either direction from the +y attachment site. The transduced host DNA enters the recipient chromosome by recombinational events which give cotransduction at greater frequency for more closely linked markers, supporting the picture that a long segment of bacterial DNA is transferred intact. The apparent high efficiency of pyr-trp integration depends upon the presence in the recipient chromosome of att for +U as well as

Addre5s to which proofs should be sent. J P GRATIA,Laboratory of Mlcroblology, Universit6 Lhre de Bruxelles, 115, Boulevard de Waterloo, 1000 Brussels, Belgium

Genetlcs 84: 663-674 December, 1976 664 J-P. GRATIA a resident prophage at that site. Furthermore, abut 1 % of the trp+ transductants in a lysogenic recipient show substitution of the prophage h marker by the h of the phi gamma transducing phage. Therefore the transducing DNA includes a small amount of DNA from the $7 vector, DNA adjacent to att on the phage genome. Thus the packaging of DNA in transducing particles is initiated within the phage genome near att (not a unique site) and depends upon phage interac- tion with host att to be extended into the host chromosome.

MATERIALS AND METHODS

1) Bacterid strains (Table 1). A series of strains were constructed with various combinations of the following markers: cysB, galU, purB, pyrF, tonB, trpA, trpB, trpE. The tonB locus was marked by a stable mutation, bXO, causing resistance to a series of colicins (B,I,M,V) and partial resistance to phage $80 (GRATIA1967). Deletions of tonB included trp and sometimes atPo. In lysogens, deletions sometimes extended to the proximal end (pB') of the $y or *80 prophage or further into the essential phage genome. Non-defective deletion lysogens were prepared by lysogenizing deletion mutants still carrying an at180 site. 2) . Two phage strains were used: (a) $yref, a phage clone reisolated after centrifugation in a CsCl gradient of a crude lysate of $y grown on CA161 (CALBERG-BACQet al. 1976). (b) a hybrid, $yhy2, formed by recombination between $y and h8'J immkZ857 to give a

TABLE 1 Origin and characteristics of E. coli K12 strains used

Strain Genotype Function' Reference and/or source

CA1 61 thi, SUII Donor S. BRENNERvia R. THOMAS 1485NF trpA, trpB; his, suf Sensitive indicator; N. FRANKLIN Recipient PA2.35.19 trpA; thi, argA, purB Recipient R. LAVALLE T122.2 trpE, cysB Recipient GRATIA1973 GT02.3 trpA, PYrF Recipient derived from 67D1 (P. FREDERICQ) GT04.2 trpE, cysB, pyrF Recipient or Donor This work+ GT06.2 bX0 Donor This work+ W3102 G9 trpB61, b80 Recipient GRATIA1967 K3 102 G35 (tonB,trpABC) del Recipient GRATIA1973 H17 galU Recipient J. SHAPIRO H4 galU; (att, tonB, trpABCD) del Recipient This work; TonB- Trp- H5 galU; (tonB,trpAB) del Recipient derivatives of HI 7 L84 (pB', tonB, trpABCD) del Recipient GRATIA1971 Md 19 (606,h,pB',tonB,trpABCDE)del Recipient GRATIA1975 1018 r- m- $ Donor CH. COLSON T128.2 trpE, cysB, pyrF; r- m- $; (680) Recipient This work Cla r- m- Sensitive indicator G. BERTANI for phage / r- m- (derivative of E. coli C)

* Donors are str-s and recipients are str-r, either originally or after derivation of resistant clones. +Recombinants obtained after transduction by $y (series GT). $1018 is a methionine-requiring mutant at an undetermined locus, as also is T128.2 which derives from it by PI-transduction (from GT04.2 to a Trp- deletion mutant of 1018, lysogenic for $80). TRANSDUCTION BY q5-y 665 phage with host range (h) of $y, immunity (imm) of 1 and the transducing properties of $7 (GRATIA1973). Lysates of $yhy2 obtained by heat induction of lysogens were usually found to contain a normal amount of transducing particles but relatively few infective units. 3) Media, phrrge plating and transduction experiments. Experimental conditions have been described previously (GRATIA1973). In preparation for phage adsorption, exponentially growing bacteria of the recipient strain were centrifuged and resuspended in TI-buffer supplemented with 20% LB broth. Small amounts of thick suspensions (0.5 - 1 x IO9 viable cells) were mixed with phage particles at a low multiplicity of infection for 30 min. at 37" and the mix- tures were then plated on Davis-Mingioli agar and incubated for 2-3 days at 37"-38". All experiments were performed under conditions of linearity, such that increasing input of phage resulted in a corresponding increase in number of Trpf colonies formed. The recipient was usually a $80-lysogen and this prophage was not displaced as a consequence of the transduction. (In some experiments in which transductants were obtained at a low rate, precautions were taken to avoid superinfection with the numerous surrounding infectious particles).

1. Euidence for linked transduction of markers located on the same side of attRo 47 tranduces markers located on either side of the prophage attachment site separately: trp on the right side and, less efficiently, galU on the left side of att8O (Figure la; Table 2, lines 1-3). With reference to markers located at some dis- tance from att80,purB and pyrF, only the latter was transduced with a frequency comparable to but somewhat lower than trp (lines 5,9). Joint transduction was observed between trp, cysB and pyrF at a characteristic frequency in all experiments (Table 2, lines 10-12). Cotransduction could not have resulted from multiple transductions of separate segments by distinct par- ticles because the multiplicity of infection was very low (1 particle for 100 or 1000 viable cells). This fact was definitely demonstrated by using mixtures of

TABLE 2 Transduction rate for nutritional markers selected separately or conjointly

Approx. Relative m.v.1. Transduction transduction Recipient Selection (pfu/v.c.)' rate rate+ (%) 1 H5 ($80h) trpAB+ 10-3 2.4 x 10-4 100 2 galU+ 10-2 1.6 x 10-5 7 3 trpf galU+ 10-1 < 10-7 0 4 PA2.35.39 (@80) trpAf 10-3 4.7 x 10~4 100 5 purBf 10-2 < 10-6 0 6 trpf pur+ 10-1 < 10-7 0 7 GT04.2 ($80) trpEf 10-3 6.1 x 10-4 100 8 cysBf 1003 4.9 x 10-4 80 9 PYrF+ 10-3 2.3 x 10-4 38 10 trpf cysf i 0-3 2.2 x 10-4 36 11 cysf pyrf 10-3 1.3 x 10-4 21 I2 trpf cysf pyrf 10-2 3.1 X 10-5 5

~~ ~ * Ratio of the phage input (pfu, plaque-forming unit of @y grown lytically on CA161) to the bacterial density (v.c., viable cell). j- The number of transductants of any type was referred to the total number of trp+ colonies. 666 J-P. GRATIA particles carrying separate segments. [In these experiments, to be reported else- where (GRATIA,unpublished), the donor strains were chosen so that the trans- duced segment was partly deleted either on the right side of trp ( lacking cysB-pyrF) or between cys and attao(deletion of the segment tonB-trpABCD). As a result, the markers trpf and cysBf-pyrFf had to be carried by different particles. At a multiplicity of infection of 0.1-1, double transductants were not observed.] Analysis of co-transduction frequencies for the markers trpE, cysB, pyrF, and also tonB (or b80) show a characteristic distribution of recombinant clones. as expected for Rec-dependent recombination requiring an even number of cross- over events (Table 3).The results of such an analysis suggests that most or all of the transducing segments include the whole set of genes from attS0to pyrF. To check this important fact more accurately, we compared the cotransduction from frequencies observed in several different experiments (Table 4). We knew that that bacterial part of the transducing segment originates from atPo;therefore no variation is expected at the left end of the host DNA segment. If the distal part of the transduced segment were absent in a substantial fraction of the transducing elements, one should expect a striking asymmetry in the cotransduction fre- quency for two given markers according to the selection. Such asymmetry was usually not observed. At most, in the example reported in Table 4, cotransduction between cysB and pyrF seemed somewhat more frequent when pyrF+ was selected (line 8) rather than cy&+ (line 12), as if pyrF were carried by most, not all, transducing particles. Evidence is given below for the existence of a small fraction of particles transducing a shorter bacterial segment. The results pre- sented in Table 4 show that joint transduction of two markers occurs at a rate proportional to the distance between them on the TAYLORand TROTTER(1972) map, indicating that the transduced segment consists of an intact linear array of genes (Figure 1 a, b) .

TABLE 3

Recombinants obtained from the transducing segment formed upon lytic infection of strain GT04.2 (trpE, cysB, pyrF)

Transduction Distribution of No. of Recipient rate for trpB+ tonB trpB trpE cysB pyrF* transduct. % 11100 63 42.0 01100 40 26.6 G9 11110 21 14.0 (@Oh) 3.5 x 1*4 01110 15 10.0 trpB, b80 01111 6 4.0 11111 3 2.0 11000 2 1.4 150

* Markers of the donor and recipient are designated by 1 and 0 respectively. TRANSDUCTION BY +y 667

TABLE 4 Linkage relationships between markers of the transducing segment*

~~ Analyzed Selected No. purified No. marker marker transductants transductants Cotrans3. uchon (AM): (SM) examined carrying AM AM/SM -+ trpA + 80 49 61.3 trpB+ 175 97 55.5 b80 trpE+ 153 44 28.7 cysB+ 150 24 16.0 PY~F+ 200 7 3.5 trpB+ 150 46 30.0 cysB trpE+ 351 108 30.7

-4 8 pyrF+ 374 160 42.8 9 trpA+ 97 5 5.2 10 PYrF trpB+ 346 25 7.2 11 trpE+ 337 29 8.6 12 cysB+ 460 114 24.8 4

* The recipients (cf. Table 1) were lysogenic for $80. Lytic and induced $y (or @y hy2) were used. t The arrow indicates the position of the analyzed marker (for -) on the map of the E. coli chromosome.

2. Evidence for hybrid structure of the left end of the atts0-trp-pyrFsegment In the experiments described above, the recipient strain carried a prophage +80. When the recipients were nonlysogenic, the transduction rate for trp+ was somewhat lower. Moreover, the frequency of cotransduction between the selected marker and the analyzed marker, when the latter was located on the left side of the former, especially tonB, which lies close to att80,was reduced in nonlysogenic recipients (Table 5).

TABLE 5 Differential efficiency of transduction with lysogenic and nonlysogenic recipients

Selected Analyzed marker Transduction marker Cotransduction Recipient (SM) Prophage.- rate/PFU* (AM) (AM-SM) ‘2, G9 trpB + $80h 7.4 x 1e4 tonB+ 97/175 55.5 (trpB, b80) - 4.7 x 10-4 tonB+ 5/80 6.3

T122.2 cysB+ $83 4.8 x le4 trpE+ 84/207 40.5 (trpE, CYSW ._ 3.6 x 1e4 trpE+ 60/208 28.8 (234/1190 19.7)t

* Particles of @yformed after lytic infection of CA161. t Result reported previously (GRATIA1973). 668 J-P.GRATIA The reduced efficiency of transduction in the neighborhood of attBoin recipi- ents with no prophage at that site was even more apparent when a tonB-trp de- letion restricted the homology between the transducing segment and the recipi- ent chromosome. As shown in Table 6, the transduction rate of the wild-type allele trp+ into a deletion mutant was almost normal when the recipient was carrying 480 (lines 3, IO) or the related hybrid prophage hh immB0with the same chromosomal location (line 5). On the contrary, the transduction rate for trp+ was considerably decreased when the same recipient carried no prophage (lines 4, 11 ) or carried a phage related to 480 but located at attXinstead of atPo (line 6). When (tonB-trp)del, gall7 recipients were used, transduction of gaZU+ was as efficient as a trp+ galU recipients, and independent of or less dependent on the presence of a prophage in attBo,presumably because genetic homology is not affected on that side. Transduction was examined with two types of deletion mutants as recipients: deletions extending (a) close to the prophage; or (b) into the prophage itself, affecting or not affecting essential genes (Table 7). When the recipients carried a complete prophage, transductants were formed at a nearly normal rate for the segment tonB-trp and retained the markers h and imm of the recipient prophage (line 2). In some experiments, however, the transductants were less frequent and included a few recombinant inheriting the h marker of +y (line 4). As we shall see now, these infrequent recombinants had received a different transducing segment. When the recipient carried a deletion extending into the prophage, the

TABLE 6 Transduction of trp+ and galU+ in deletion mutants; helper effect of a prophage in attso

Relative transduction rate (%) * Recipient Mutation Pruphage trp+ gnlU+ 1 1485NF trpA, -B +go 100 - 2 - 64 - 3 G35 (tonB, trp) del @Oh 58 - 4 - 0,03 - 5 hX attso immso 71 - 6 hX attX imms0 0,2 - 7 HI7 gQlU 8

9 H4 galU, - 0 81 (att, tonB, trp) del 10 H5 galU, +80h 81 71 11 (tonB, trp) del - 3 68

* The donor strain was CA161 and the vector cy. In each column, the number of transductants was referred to the number of transductants of the corresponding type derived from the lysogenic recipient carrying no deletion (line 1 for trp+ and line 7 for galU+). Various lysates were com- pared (cy and cyhy2, either induced or lytic); when sometimes they showed different efficiencies of transduction to a given recipient, they gave rise to the same relative transduction rate, which appears then as a characteristic of the recipient. TRANSDUCTION BY +y 669 TABLE 7

Differential efficiency of transduction of trp+ in deletion mutants carrying a prophage, either normal (G35, H5) or defective (L84, Mdi9) ; recombination of prophage markers

~ ~ ~ ~ Transduction No. recomb. Prophage Relative Recipient rate for tr&' analyzed markers frequency+

1 1485 (~80) 3.0 x 10-3 100 h80 imm80 100 2 G35 (+%Oh) 2.2 x 10-3 50 h80 imm80 73 3 H5 (@Oh) 5 x 10~4 46 ha0 imm80 17 4 H5 (~80h) 5xlO-i 2 hY imm80 0.7 5 L84$ 1.7 x 10-5 5% hY immao 0.6 6 Mdl9 2.2 x 10-5 5% hy immao 0.7

* Lysate of induced 9yhy2 in CA161. 4 Frequency referred to that of the normal case (line 1: 100%) on the basis of the transduction rate. $ L84 is carrying a barely deleted prophage following a deletion event affecting excision but not essential genes. s Part of the unpurified transductants were found to segregate cells cured of their prophage. transduction rate dropped a hundredfold. This was observed with any deletion lysogen, including those carrying a barely deleted prophage 480, as in strain L84 (line 5), or $7 itself (unpublished). Transductants were still true recombinants as shown by the absence of TonB- segregants [colonies resistant to phage TI were spontaneous mutants mostly of the TonA- type]. They all carried a recom- binant prophage hXimm80 and resulted therefore from a crossover beyond h*. The occurrence of such transductants was infrequent (lines 4, 5, 6 : 0.7% of the normal frequency). Transductants that inherited the marker hr were examined in cases where the donor chromosome was marked by mutations on the right side of the deletion and a striking difference appeared: only a few received the marker cysB and none inherited pyrF (Table 8). Fractions of phage preparations ob-

TABLE 8 Recombination pattern of a hybrid transducing segment in a defectiue deletion lysogen*

Segregation of markers: No. transductants analyzed+ Prophage Bacterium Total Per class CI h tonB trpE cysB pyrF

93 0 1 1 1 0 0 99 2 0 1 1 1 1 0 3 - - 1 1 0 0 1 - - 1 1 1 0

* Strain Md19, with a deletion covering trpABCDE-tonB-att-h. The donor (GT04.2) was marked by mutations in trpE, cysB and pyrF. The vector was the induced Gyhy2. )One purified clone per transductant was analyzed; among the lysogenic types, cure of the prophage was sometimes observed after reisolation or subculture. $ The transduced markers are designated by 1. Repeated reisolation or subculture reveals spontaneous curing of the prophage in some of these transductants, probably due to a genetic defect of the recombinant prophage, since the phage produced by such strains was still able to lysogenize other hosts but, again, was not maintained indefinitely in them. 670 J-P. GRATIA tained after centrifugation in a CsCl gradient+ have been compared in order to see if the rare particles which are carrying the trp-tonB-hr segment differ in den- sity from the usual transducing particles; considering the fluctuations in trans- ducing frequencies for each of the 2 types, no clear dissimilarity in the DNA content was observed. These observations indicate that the transducing segment of most particles includes a short part of the prophage, just enough to allow normal pairing and crossing over in lysogenic recipients (Figure 1 b, c, d) . Use of deletion lysogens as recipients revealed that infrequent transductants can be formed following recombination with another transducing segment of the same size but extending beyond h in the prophage and ending around cysB in the bacterial genome (Fig- ure le). No difference has been detected between lysates prepared by induction of the lysogenic donor strain or by lytic growth on nonlysogens.

3. Effect of the restriction-modification (RM) system of E. coli K12 on trans- duction of the tq-pyrF segment from modificationless strains. In order to check on the integrity of the transduced host DNA when it enters the recipient bacteria, we used a modificationless strain as donor. Previous ob servations (GRATIA1971; MURRAYand BRAMMAR1973) indicate the presence of a restriction target in the trp region. If the transducing DNA were carrying such site susceptible to restriction, transduction as well as transfer by conjuga- tion (ARBERand MORSE1965) would be affected in a K12 recipient, especially cotransduction of markers located on either side of the cleavage site. Although transduction of each selected marker -trpE+, cysB+ or pyrF+ - occurred between two modification- (r-m-) strains at the rate characteristic of the two r+m+ strains studied up to now, transduction from an m- donor to an r+m+ recipient was considerably reduced (Table 9). Restriction impaired transduction of cys+ and pyr+ even more than transduction of trp+. Also, co- transduction frequencies drastically decreased between trp+ and cys+ or pyrf . Yet, cotransduction of cys+ and pyr+ appeared to be unaffected (last column, line 2).

TABLE 9 Effect of the K-restriction system on the transducing segment formed after lytic infection of KI2 r- m- strain 1018

Transduction rate for Cotransduction hetweent Recipient * irpEf cysB+ pyrF+ trpE-cysB irpE-pyrF cysB-pyrF T128.2 6.5xlOW 5.4x1C4 2.7x1C4 225/492 8/92 108/4.00 (rm-) (45.8%) (8.7%) (27%) GT04.2(@80) 7.8~10-6 2.3xlG-6 +1.4xlQ-6 4/193 0/173 23/81 (r+ m+) (R=83) (R=235) (R=L200)+ (2.1%) (<0.6%) (28.4%)

* Both recipients carry mutations in trpE, cysB and pyrF and are lysogenic for @80. t R = ratio E.O.T. on rm-/E.O.T. on rf m+. $ Number of cotransductants among transductants selected for one marker.

t The author thanks DR. FRAN~OISESqum and S4~4KOZMA who performed these centrifugations. TRANSDUCTION BY (67 671

DISCUSSION

The means by which (6y packages host DNA is obscure. One approach to un- derstanding the mechanism involves analysis of the genetic content of the trans- ducing particles. At first, we attempted to identify the transducible genes and to characterize the bacterial segment which can be packaged, especially with regard to its length and structure. Markers located on the left side (gaZU) and, more efficiently, markers located on the right side (trp) of atts0are transduced separately. Genes between atPo and pyrF are transduced conjointly and belong to a linear segment. It is possible that the distal marker pyrF is located in the vicinity of the right end, since its incorporation, requiring a crossover to its right, occurs less efficiently than recombination of internal markers. The fact that the transducing segment is relatively long and seems to have definite ends is significant in connection with the biophysical characterization of the virion. (6~has a unique double-stranded DNA molecule 13-14p long with a molecular weight of 28 X IO6 daltons (CAL- BERG-BACQet al. 1976), like (680 (YAMAGISHI,YOSHIZAKO and SATO1966). But lysates of (67, unlike the LFT lysates of phages A and (680, have a high transducing activity due to a dense and homogeneous population of particles, the DNA of which is uniform and very similar to the normal phage DNA in length and in overall base composition ( CALBERG-BACQet al. 1976). According to the map drawn by TAYLORand TROTTER(1972), recalibrated by BACHMAN,Low and TAYLOR(1976) , the distance between atPo and pyrF seems to correspond to 0.8-1.0% of the total chromosome. If this estimate is correct, and if the E. cdi chromosome measures 11 OOp (CAIRNS1963; BLEECKEN,STROH- BACH and SARFERT1966), the distance between att8O and pyrF would be 9-1 1p long. The bacterial segment of the transducing particle should be somewhat longer, just enough to allow crossing over on the right side of pyrF. The transducing DNA of dy, which, like the normal phage genome, would measure 13-14 p is likely to be long enough to include a small amount of phage DNA. In agreement with this expectation, we obtained genetic evidence that a short part of the (6y chromosome is joined to the bacterial segment. Results of transduction experiments using deletion mutants as recipients are interpreted to indicate that the trp-pyrF transducing segment carries DNA from the left side of tonB, in particular the +y prophage end homologous to the proximal end of the recipient’s prophage (Figure 1) . This homologous stmcture includes the hybrid sequence pB’ and some adjacent part of the (67 genome. The latter, however, is probably too short to allow crossing over within the prophage on the left side of a terminal deletion whether the resident prophage is completely homologous ((6y) or not ((680). Exceptionally, a few transductants indicated the presence of a longer prophage segment including hr. In other transducing systems, diff erent degrees of hybridization between bac- terial and phage DNA have been reported. On the one hand, defective trans- ducing particles of 480 (or A) contain a short bacterial segment and a reciprocally large prophage segment which includes the cohesive ends needed to circularize 6 72 J-P. GRATIA trP 90 A B CD E cysB pyrF a 1 < (27min)*0,7min (gp?) *

b

C

'DB' d

FIGURE1.-Genetic map of Cy transducing DNA. a) Part of the E. coli chromosome around att80. b) The transduced segment located on the right side of ait80. c) Pairing and crossing-over of such segment with the recipient chromosome; pairing should be complete when the prophage is present in att80 and incomplete when the prophage is absent. d) The reduced homology between the transducing segment and the recipient chromosome carrying a deletion and no prophage lowers the probability of crossing over on the left side. e) Recombination of a hybrid prophage-bacterial segment into a defective deletion lysogen. Single line: bacterial chromosome; double line: prophage; broken line: crossing over; hatching: deletion.

DNA before integration. The amolunts of bacterial and phage DNA are, in fact, independent variables (CAMPBELL1971; GRATIA1975). On the other hand, any transducing DNA of the generalized transducer P22 includes a large segment of bacterial genes and a contribution of only abut 10% of newly synthetisized DNA, presumably of phage origin (SCHMIEGER1968; SCHMIEGERand BACKHAUS 1973). Between such extreme types, exceptional forms of transducing particles of these phages may resemble the one described here. They result from excision- TRANSDUCTION BY +y 673 defective lysogens of either X (LITTLEand GOTTESMAN1971; STERNBERGand WEISBERG1975) or P22 (SMITH1968). In both cases the transducing segments detached from the integrated prophage are linear and hybrid; transduction occurs by substitution of host DNA for homologous recipient DNA. In A, detachment is facilitated by a sequence-specific cutting activity (Ter) at the cohesive end site (cos). For a comparable phenomenon to occur with 47, one must assume that (1) the infective phage genome is inserted into the hoist chromosome; (2) this event is frequent but transient, not followed by repression and then derepres- sion; (3) detachment is promoted by some specific cutting activity of the Ter type (a cos site has not yet been looked for in this phage) ; (4) a part of the detached segment is packaged having the same length as the normal phage genome but with different end-points, preferentially near the att site. Until such assumptions receive some experimental support, we propose an alternative hypothesis based on the finding of CALBERG-BACQ et al. (1976) that, from its melting temperature and its buoyant density in CsCl, +y DNA has a base composition very close to that of DNA. One can speculate that both would pair to some extent, depending on the degree of sequence homology, as soon as they attach to each other at the atPo site. Let us consider the circular form of +y DNA activated by the Int function, either induced and going to be excised or infective and going to be inserted; the bacterial DNA surrounds it; either a loop is detached by a cut at two contiguous but unlinked points, or copies are formed along an in situ template. Such fragments or copies (a) would include primarily bacterial DNA and some hybrid DNA around the attachment point; (b) would have the same length as the phage genome; and (c) would be linear, ending on one side in a short phage DNA segment and on the ather side beyond the most distal transduced gene. This model has the advantage of simplicity; it does not involve integration of the infective phage genome and cutting at different points in several steps, a complex mechanism which seems unlikely to occur at a relatively high frequency. The author is indebted to C. COLSONand R. THOMASfor gifts of strains and for critical reading of the manuscript, and to J. HOSTENSfor valuable technical assistance.

LITERATURE CITED ARBER,W. and M. L. MORSE,1965 Host specificity of DNA produced by Escherichia coli. VI. Effects on . 51 : 137-148. BACHMAN,B. J., K. BROOKSLow and A. L. TAYLOR,1976 Recalibrated linkage map of Escher- ichia coZi K12. Bacteriol Rev. 40: 116-167. BLEECKEN,S. G. STROHBACHand E. SARFERT,1966 Autoradiography of bacterial chromosomes. Z. Allgem. Mikrobiol. 6: 121. CAIRNS,J., 1963 The chromosome of Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 28: 43. CALBERG-BACQ,C. M., F. SIQUET-DESCANS,S. KOZMA and J. P. GRATIA,1976 $y: a coliphage related to, but distinct from the $80 virion. Intervirology 6: 98-107. CAMPBELL,A. M., 1971 Genetic structure. pp. 13-44. In The Bacteriophage Lambda (Edited by A. D. HERSHEY.)(Cold Spring Harbor Laboratory). 6 74 J-P. GRATIA GRATIA,J. P., 1967 Variations du taux d‘adsorption du bactkriophage $80 en rapport avec la resistance ti la colicine B. Ant. v. Leeuwenhoek 33: 153-158. __, 1971 Dklbtion et substitution de sites de restriction dans un phage hybride lambda-80. Ann. Inst. Pasteur 121 : 13-22. --, 1973 Coliphage $y, a novel type of specialized transducer. Mol Gen. Genet. 124: 157-166. --, 1975 Etude des mkcanismes de la transduction par le bac- tkriophage $80 d’Escherichiacoli. Arch. Biol. (Bruxelles) 86: 1-44. LITTLE,J. W. and M. GOTTESMAN,1971 Defective lambda particles whose DNA carries only a single cohesive end. pp. 371-394. In: The Bacteriophage Lambda, (Edited by A. D. HERSHEY.)(Cold Spring Harbor Laboratory). MURRAY,N. E. and W. J. BRAMMAR,1973 The trpE gene of Escherichia coli K contains a recognition sequence for the K-restriction system. J. Mol. Biol. 77: 615-624. SCHMIEGER,H., 1968 Die molekulare Struktur transduzierender Partikel beim Salmonella- Phagen P22. I. Dichte gradienten-Untersuchungen an intakten Phagen. Mol. gen. Genet. 102: 336-347. SCHMIEGER,H. and H. BACKHAUS,1973 The origin of DNA in transducing particles in P22- mutants with increased transduction frequencies (HT-mutants) Mol. gen. Genet. 120: 181-190. SMITH,H. O., 1968 Defective phage formation by lysogens of integration deficient phage P22 mutants. 34: 203-223. STERNBERGN. and R. WEISBERG,1975 Packaging of prophage and host DNA by coliphage A. Nature 256: 97-103. TAYLOR,A. L. and C. D. TROTTER,1972 Linkage map of Escherichia coli strain K12. Bacteriol. Rev. 36: 504-524. YAMAGISHI,H., F. YOSHIZAKOand K. SATO,1966 Characteristics of DNA molecules extracted from bacteriophages $80 and $80 ptl. Virology 30: 29-35. Corresponding editor: I. P. CRAWFORD