Fine Structure Genetic and Physical Map of the Gene 3 to 10 Region of the Bacteriophage P 22 Chromosome Shenvood Casjens , ’ Kathryn Eppler ?

The mechanism by which dsDNA is packaged by viruses is not yet understood in any system. Bacteriophage P22 has been a productive system in which to study the molecular genetics of virus particle assembly and DNA packaging. Only five phage encoded proteins, the products of genes 3, 2, I , 8 and 5, are required for packaging the virus chromosome inside the coat protein shell. We report here the construction of a detailed genetic and physical map of these genes, the neighboring gene 4 and a portion of gene IO, in which 289 conditional lethal amber, opal, temperature sensitive and cold sensitive mutations are mapped into 44 small (several hundred base pair) intervals of known sequence. Knowledge of missense mutant phenotypes and information on the location of these mutations allows us to begin the assignment of partial protein functions to portions of these genes. The map and mapping strains will be of use in the further genetic dissection of the P22 DNA packaging and prohead assembly processes. T HE life cycle of the Salmonella typhimurium lysogenic bacteriophage P22 has been the subject of extensive genetic and biochemical investigations that have made it one of the most well understood viruses (SUSSKIND and BOTSTEIN 1978; POTEETE 1988). One of the interesting but incompletely understood aspects of P22 growth is the packaging of the dsDNA chromosome within the coat protein shell. During virion assembly the 43,400-bp phage chromosome is packaged by a complex series of reactions in which (1) phage concatemeric DNA is recognized as the proper substrate for packaging, (2) DNA enters precursor particles called proheads, (3) chromosome length DNA molecules are nucleolytically cleaved from precursor DNA, (4) a major precursor particle protein (scaffolding protein) leaves the structure and remains intact to reassemble into new proheads, ( 5 ) the coat protein shell expands about 11% in radius, (6) ATP is cleaved, and (7) the products of genes 2 and 3 act but are not found in the completed virion (reviewed by CASJENS 1989). The gene 3 protein forms a complex with gp2’ (POTEETE and BOTSTEIN 1979), and it is at least partially responsible for recognition of phage DNA (RAJ, RAJ and SCHMIEGER 1974; JACKSON, LASKI and ANDRES 1982; CASJENS et al. 1987). Both the gene 2 and 3 proteins are required for cleavage of the DNA concatemer (LASKI andJAcKSON 1982). DNA insertion into the prohead is thought to begin at a site called pac and proceed unidirectionally from that point until the prohead is filled with ’ To whom correspondence should be addressed. ‘Current address: Natural Product Sciences, Inc., 420 Chipeta Way, Salt ’ Abbreviations used g p X , the gene product of cistron X . Lake City, Utah 84108. Genetics 147: 637-647 (April, 1991) DNA (103.8% of the sequence), at which point a “headful” cleavage is made in the DNA, freeing the packaged DNA from the concatemer. Subsequent packaging events start from the concatemer end created by the previous event, resulting in processive packaging series typically 2.5 to 5 events long (TYE, CHAN and BOTSTEIN 1974; JACKSON, JACKSON and DEANS 1978; WEAVER and LEVINE 1978; KUFER, BACKHAUS and SCHMIEGER 1982; CASJENS and HUANG 1982; ADAMS, HAYDEN and CASJENS 1983; BACKHAUS 1985; CASJENS and HAYDEN 1988). This is a common replication/packaging strategy for bacteriophages, and in addition, the iridoviruses of animals appear to use a similar strategy (reviewed by CASJENS 1989). The genes, 3, 2, 1, 8 and 5, that encode the five P22 proteins required for DNA packaging, lie in a contiguous cluster in the late operon (BOTSTEIN, CHAN and WADDELL 1972; EPPLER et al . 1991). The transient function of the gene 3 and 2 proteins in DNA packaging was mentioned above. The gene 1 protein is thought to function as a “portal” through which DNA enters the prohead (BAZINET et al. 1988). Scaffolding protein is encoded by gene 8 and forms the internal core of proheads, which leaves the structure during DNA packaging (KING and CASJENS 1974). Coat protein, encoded by gene 5, forms the outside shell of proheads and completed virions (KING, LENK and BOTSTEIN 1973; EARNSHAW, CASJENS and HARRISON 1976). In order to further our understanding of the genes and proteins that participate in the DNA packaging process, we report here the construction of a detailed genetic and physical map of the region of the bacteriophage P22 chromosome that contains these genes as well as a nonessential 638 S. Casjens et al. open reading frame ORF109, gene 4 and a portion of gene 10 (EPPLER et al. 1991). The products of the latter two genes stabilize the DNA within the head after it is inserted into the coat protein shell (STRAUSS and KING 1984). A low resolution genetic/physical map of the gene 3 to 10 region of the P22 chromosome has been previously constructed (GOUGH and LEVINE 1968; CHAN and BOTSTEIN 1972; BOTSTEIN, CHAN and WADDELL 1972; R U T I L A ~ ~ ~ JACKSON 1981; WYCKOFF and CASJENS 1985; RIGGS and BOTSTEIN 1987), and the proteins encoded by each of the genes have been identified by SDS-polyacrylamide gel electrophoresis (BOTSTEIN, WADDELL and KING 1973; KING and CASJENS 1974; POTEETE and KING 1977; YOUDERIAN and SUSSKIND 1980). We have recently completed the nucleotide sequence and determined the precise gene placement in this region of the P22 chromosome (EPPLER et al. 1991). A large number of conditional lethal mutations have been previously isolated for P22. We present here the use of these mutations and the nucleotide sequence information to construct a very detailed genetic/physical map of this region. One eventual goal is the correlation of partial functions of the proteins involved in DNA packaging with particular portions or domains of the proteins. MATERIALS AND METHODS Bacteria, phage and plasmids: Salmonella typhimurium DB7000 (sup', leu am414) (SUSSKIND, WRIGHT and BOTSTEIN 1974) was used as host for amber+ P22 growth. The closely related amber suppressing strains DB7 154 supDl O(Ser), DB7 155 supE20(Gln), DB7 156 supFPO(Tyr) and DB7 157 supJ60(Leu) (WINSTON, BOTSTEIN, and MILLER 1979) were used for growth of P22 amber mutants and to test the suppression patterns of the amber mutants. DB109 was used to propagate ug phage mutants (CHAN and BOTSTEIN 19'12). All Salmonella strains were from the collection of D. Botstein. Escherichia coli strain MC1061 (CASADABAN, CHOW and COHEN 1980; RALEIGH et al. 1988) was used to carry plasmids. M 13 phage vectors and their host are described by YANISCH-PERRON, VIERA and MESSING (1985). The isolation and description of the P22 conditional lethal mutations used in the construction of the genetic/physical map are described in the references that follow. Allele names beginning with H, N or U were isolated by hydroxylamine, nitrosoguanidine, or UV mutagenesis, respectively (unless otherwise indicated, the mutants were from the collection of D. BOTSTEIN and were gifts from D. BOTSTEIN and A. POTEETE): ugH1-ugH99 and amH200-umH299 (LEW and ROTH 1970; ug mutations are suppressed by UGA or opal suppressors and am mutations are suppressed by UAG or amber suppressors); amH l-amH 100 (KOLSTAD and PRELL 1969; gifts from H. PRELL and D. BOTSTEIN); amN1-amNlOO and amH300-amH400 (BOTSTEIN, CHAN and WAD DELL 1972); amNl OO-amN199 (isolated in the M. LEVINE laboratory-see BOTSTEIN, CHAN and WADDELL 1972); amU200-amU243 (gift from J. KING and P. PREVELIGE, unpublished; SMITH, BERGET and KING 1980); amH1000amH1399 (POTEETE and KING 1977; RIGGS and BOTSTEIN 1987); amH 1400-amH 1499 (gift from M. SUSSKIND, unpublished); tsl.1-ts26.1 (GOWGH and LEVINE 1968); tsN1-tsN99 (isolated in the M. LEVINE laboratory-see BOTSTEIN, CHAN and WADDELL 1972); ts su(amUT34)5, ts su(amUT7l)l, ts su(amY232)ll and tsU172, (gifts from J. KING and P. PREVELIGE, unpublished; BAZINET and KING 1988); tsNl00N 199, csH1-H199 and other ts and cs mutations (JARVIK and BOTSTEIN 1973 and 1975;JARv1~ 1975; J. JARVICK and D. BOTSTEIN, unpublished); the phage L amber mutations were isolated by J. SOSKA (gifts of J. SOSKA and W. BODE; KARLOVSKY et al. 1984). The plasmids constructed for use in the creation of the genetic/physical map are described in Table 1 , and their P22 DNA inserts are shown schematically in Figure 1 . The locations of the various restriction sites can be found in CASJENS et al. (1 983), EPPLER et al. (1 99 1) and the references therein. Deletion mapping strains f223 through f236 are described by RIGGS and BOTSTEIN ( 1 987). DNA manipulations: DNA isolation, cleavage by nucleases, end blunting, ligation and transformation were performed as previously described (WYCKOFF and CASJENS 1985; WYCKOFF et al. 1986), except that plasmids were moved into Salmonella by electroporation with a Bio-Rad Gene Pulser (25 pF, 1.25 kV, 800 Q , with 0.2-cm cuvettes containing 1 rl of DNA solution and 40 @I of cell suspension [ 1 X 10" cells/ml in 3 mM K P 0 4 , pH 7.4, 272 mM sucrose, 15% glycerol]). Typically a few hundred transformants of DB7000 were obtained per pg of plasmid DNA from an E. coli mini-lysate.