STUDIES ON THE MECHANISM OF TRANSDUCTION BY BACTERIOPHAGE +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 prophage attachment site, e.g., on the right side: att80-to&- trpABCDE-cysB-pryF. The att end includes a variable amount of phage genome, 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 bacteria 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) Bacteriophages. 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 (plasmid 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.