Mutations in Coliphage P1 Affecting Host Cell Lysis
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JOURNAL OF VIROLOGY, Aug. 1980, p. 519-530 Vol. 35, No. 2 0022-538X/80/08-0519/12$02.00/0 Mutations in Coliphage P1 Affecting Host Cell Lysis JEAN TWEEDY WALKER* AND DONALD H. WALKER, JR. Department ofMicrobiology, University of Iowa, Iowa City, Iowa 52242 A total of 103 amber mutants of coliphage P1 were tested for lysis of nonper- missive cells. Of these, 83 caused cell lysis at the normal lysis time and have defects in particle morphogenesis. Five amber mutants, with mutations in the same gene (gene 2), caused premature lysis and may have a defect in a lysis regulator. Fifteen amber mutants were unable to cause cell lysis. Artificially lysed cells infected with five of these mutants produced viable phage particles, and phage particles were seen in thin sections of unlysed, infected cells. However, phage production by these mutants was not continued after the normal lysis time. We conclude that the defect of these five mutants is in a lysis function. The five mutations were found to be in the same gene (designated gene 17). The remaining 10 amber mutants, whose mutations were found to be in the same gene (gene 10), were also unable to cause cell lysis. They differed from those in gene 17 in that no viable phage particles were produced from artificially lysed cells, and no phage particles were seen in thin sections of unlysed, infected cells. We conclude that the gene 10 mutants cannot synthesize late proteins, and it is possible that gene 10 may code for a regulator of late gene expression for P1. A genetic map for P1 (Fig. 1), consisting of 113 mutants for their ability to lyse sup0 cells. Those amber mutations, has been established (31) us- mutants that lyse supo cells would probably have ing deletion prophages, two-factor spot tests, a defect in late genes involved in morphogenesis, and three-factor crosses. The two-factor crosses whereas those that do not lyse sup' cells would permitted assignment of the mutations into 10 probably have an early gene defect or a defect linear arrays or linkage clusters, I through X, in a late gene determining a lysis function. with mutations in cluster X behaving as two subgroups, X-1 and X-2. However, cistron des- MATERIALS AND METHODS ignations, apart from those originally assigned by Scott (22), were not assigned because com- Bacterial and phage strains. Bacterial strains tests in are described in Table 1. Phage strains are Plvir (the plementation liquid frequently gave am- supervirulent mutant Plkcvie of Sarkar which grows biguous results. on strains lysogenic for P1; 22); Plvir amber mutants With other phages, amber mutants can be (Fig. 1) 1, 2,4, 6, 7, 11, 13, 14, 16, 17, 19, and 21 of Scott divided into two major classes depending on (22) and 91 vir amber mutants of Walker and Walker whether they have a gene defective in a function (30) (for simplicity these mutants will be referred to which is required early or one which is required as aml, etc., without the vir designation); T6 and late in phage particle development ("early" and AcI857Sam7 (gifts from M. Howe); and AcI857Ram5 "late" genes). Late genes are involved in particle and AcI857 (gifts from M. Feiss). morphogenesis (e.g., production of proteins Media, phage assays, and preparation of which become structural components ofthe par- phage stocks. These were the same as described previously (30), except that L-broth (1% Trypticase ticle) and cell lysis (e.g., genes R and S in A; 9, [BBL Microbiology Systems], 0.5% yeast extract 10). Mutants having a defect in late genes in- [Difco], 1% NaCl, 0.1% glucose, and pH adjusted to 7.4 volved in particle morphogenesis can lyse the with 1 N NaOH) was used instead of WLB. In exper- host cell under nonpermissive (sup0) conditions, iments where cells were infected with A wild-type or whereas those having a defect in a lysis function mutant phages, cells were grown in L-broth containing cannot. Early genes are involved in regulation of 0.1% maltose and 10 mM MgSO4 instead of 0.1% transcription, DNA synthesis, and recombina- glucose. Streptomycin (streptomycin sulfate; Pfizer tion (e.g., genes N, 0, P, and Q in A). In addition, Laboratories) was used at 10 iLg/ml; spectinomycin (spectinomycin hydrochloride; Upjohn) was used at mutations in genes such as N, 0, P, and Q do 40 ug/ml; and tetracycline (tetracycline hydrochloride; not allow synthesis of any of the late gene prod- Lederle) was used at 10,Ig/ml. ucts of A, including those effecting lysis. There- Lysis of phage-infected cells. At 0 min the de- fore, to help determine which P1 amber mutants sired amber mutant was added (at a multiplicity of are defective in early genes and which are defec- infection [MOI] of 4 to 6 except where stated other- tive in late genes, we have tested 103 P1 amber wise) to a log-phase L-broth culture of Shigella dysen- 519 520 WALKER AND WALKER J. VIROL. LINKAGE I I CLUSTER m CISTRON CB 1 c3 c2 4 2 3 wOir .., ...I-.I-I. ., _ .I . _*I * - .s I r. AMBER 47141 3734 4 17357 129 7 62 117 108 23 14473 134 20 17 33 72I" 66 49 127 6 171 75 77 128 76 150 174 31 114 74 21 MUTATION 132 ifl 56 35 165 103 118 170 141 LYSIS 1NORMAL NO __j L JvI NORMALI~~~~~~~~~~~~~~~~~~~~~~~~~~155 PHENOTYPE LYSIS PREMATURE ME G Ix 1- 2 I- 1 C7 5 6 CS B 10 9 Ll \ 1*1......o.... !. _ r -.-." L% .,.. , . ., ... , 11 1(a!1 3 - L "" J 135 107 45 28 19 162 2 79 26 32 14 39 53 22 102 30 40 29 139 51 71 13 178 1 36 16 152 4664 48 43 30 115 138 5 109 27 67 116 78 42 101 151 68 11 104 54 136 58 106 Fe 61 179 110 125 160 113 142 140 70 105 131 112 III 137 175 -NORMAL IL NO -J L NORMAL-I r, LYSIS FIG. 1. Genetic map ofPl amber mutations, modified from Walker and Walker (31). Regions demarcating the 10 linkage clusters are represented by solid blocks. The cistron designations above the map line include the original 1 to 10 ofScott (22). Other designations are vir and eight c genes (22-25; J. R. Scott, unpublished data). The orientation of groups of amber mutations within parentheses or within brackets with respect to mutations outside these groups is unknown. Since this map was first published, the orientation of the group ofmutations in linkage cluster I and ofthe group ofmutations, 127 through 75, in linkage cluster IV has been established (J. R. Scott, R. Mural, and D. Vapnek, personal communication) by three-factor crosses and by rescue of am' alleles from cloned P1 DNA fragments which had previously been mapped by restriction endonuclease analysis (1). In addition, rescue tests with such cloned fragments has established that the order ofthe subgroups X-1 and X-2 and the orientation ofsubgroup X-1 is as shown (Stemnberg, as cited in reference 32). The spacing of the hash marks is not meant to indicate either genetic or physical distance. Amber mutations that are underlined denote those mutants that were not used in the experiments reported in this paper. TABLE 1. Bacterial strains the culture was diluted 2.5-fold in fresh medium con- taining 5 mM MgSO4 and incubated at 37°C with Strain desig- Relevant genotype' Source or refer- nation' ence vigorous shaking. The optical density of the culture was read at 5- to 10-min intervals at a wavelength of 600 nm in a Bausch and Lomb Spectronic 20 spectro- Sh-i5 sup0 Sh (2) a Sh-16 Sh-15 str Sh/s; (2) photometer. An uninfected culture and culture in- Sh-16supF Sh-16 supF tet M. Van Montagu fected with Plvir were included as controls. Cultures DW101 sup° (30) that did not lyse were assayed for viable cells. In DW103 DW101 supD (30) experiments where burst sizes were measured, infected DW104 DW101 str This laboratory Sh-15 or DW101 cultures were washed by filtration DW106 DW101 str tsx This laboratory after the 10-min adsorption period, to get rid of un- DW107 DWIOI str tsx spc This laboratory adsorbed phage, and progeny phage were assayed on DW304 DW103 str This laboratory sup° and sup' strains Sh-16 and or DW104 DW306 DW103 str tsx This laboratory Sh-16supF DW307 DW103 str tsx spc This laboratory and DW304. R594 supo str (3) Artificial lysis ofphage-infected cells by using MF380 R594(XcI857Aam32) M. Feiss phage T6. Cells of E. coli K-12 sup° strains DW101 or R594 were infected with amber mutants or with 'Abbreviations and genotypes: Sh-15 and Sh-16, strains of S. dysenteriae; DW strains, derivatives of E. coli K-12 sup', Plvir (MOI, -5). After 10 min at 37°C, the cells were nonpermissive for amber mutants; supD and supF, suppres- washed on a Millipore membrane filter, diluted 1:1 in sors of amber mutations; str, streptomycin resistance; spc, fresh L-broth, and incubated at 37°C until 80 min spectinomycin resistance; tet, tetracycline resistance; tsx, re- (unless stated otherwise) after infection. Cultures were sistance to phage T6. divided, and T6 was added to one sample (MOI, -500) and P1 buffer was added to the other. After incubation teriae sup° strain Sh-16 or Sh-15, or Escherichia coli at 370C for 15 min, cultures which previously had not K-12 supo strain DW101.