Molec. gen. Genet. 143, 307-309 (1976) © by Springer-Verlag 1976 Altered Cotransduction Frequencies Exhibited by I-IT-Mutants of Salmonella-Phage P22 Horst Schmieger and Horst Backhaus Genetisches Institut der Universitgt Mfinchen Summary. Phage P22-mutants with increased trans- duction ability (HT-mutants) show in comparison to wild type P22, different frequencies of cotransduction for markers on two different transducing fragments of the Salmonella chromosome. The data are inter- preted as indicating that host DNA to be packaged is cut by HT-mutants at sites different from those cut by wild-type phage, due to an altered specificity of the nuclease responsible for this step. In generalized transduction by Salmonella phage P22 different markers are transducible with widely differ- ing frequencies (Zinder, 1955; Ozeki, 1959; Schmieger, 1972). In addition Ozeki obtained experimental evi- dence that the endpoints of the transducing fragments are not distributed at random. He found that all trans- ducing particles which carry the particular markers he studied carry identical pieces of the bacterial chro- mosome. Other authors however found evidence for some heterogeneity among transducing fragments (Roth and Hartman, 1965 ; Pearce and Stocker, 1965 ; Enomoto, 1967; Kemper, 1974). Their results show that at least some markers can be transduced by more than one defined fragment. On the basis of Ozeki's arguments, Smith-Keary and Dawson (1963) calcu- lated the absolute positions of leu and ara mutant sites on the transducing DNA-fragment. For P22-mutants with highly increased transduc- tion abilities (HT-mutants) it could be demonstrated that not only are the transduction frequencies in gen- eral increased but the marker specific differences disap- pear almost completely (Schmieger, 1972). This obser- vation was interpreted as meaning that a substrate specific step during the formation of transducing parti- cles becomes less fastidious. This step could be the cutting of the bacterial DNA during the packaging process. During HT-phage maturation, therefore, excision of pieces of host DNA should occur every- where along the chromosome with similar frequencies. This hypothesis is supported by the map position of the gene responsible for HT-phenotype: it is identi- cal with gene 3 of the P22-map (Raj et al., 1975) which seems to be involved in fragmentation of the phage DNA concatemers and possibly codes for the endonuc- lease necessary for cutting (Botstein et aI., 1973). If this interpretation is correct and if the specificity of the cutting enzyme is responsible for specific endpoints of transducing DNA-fragments as well as the fre- quency with which any one fragment will be packaged, one should expect that at least some transducing frag- ments in HT-phages have different ends as compared to wild type phages. An altered position of markers relatively to the fragment ends can be tested by cotrans- duction analysis. We therefore performed cotransduction experi- ments with lysates of H5, a clear plaque mutant of P22 (wild type transduction ability) and HT12/4 (in- creased transduction ability) using markers on two different transducing fragments. Phages were grown on wild type strain LT-2. Transductions were per- formed by infecting an overnight culture of the relevant lysogenic recipient strain with H5 or HT12/4 (moi < 10). After adsorption (10 min, 37 °) appropriate dilutions were plated on M9 agar plates selective for one marker. After growth of transductant colonies the plates were replica plated on media selective for the other marker(s). Cotransduction frequencies were determined with each mutation serving as selected and unselected marker, so that linkage in both directions was examined. First we studied the transducing fragment with the markers leu + ara + . Recipient strain was SA4(P22): leuA124- araB9-, kindly provided by K.E. Sanderson. Table 1 shows the differences in cotransduction fre- quencies which depend on a) the phage lysate (H5 or HT12/4)
