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Copyright 0 1989 by the Genetics Society of America The Isolation and Sequenceof Missense and Nonsense Mutations in the Cloned Bacteriophage P22 Tailspike Protein Gene John J. Schwarz' and Peter B. Berget' Department of Biochemistry and Molecular Biology, University of Texas Medical School and Graduate School of Biomedical Sciences, Houston, Texas 77025 Manuscript received August 5, 1988 Accepted for publication December 19, 1988 ABSTRACT Twenty-seven new mutations in the structural gene for the Salmonella typhimurium bacteriophage P22 tailspike protein have been isolated, mapped using a powerful plasmid-based genetic system and their DNA sequence changes determined. Themutations were generated by hydroxylamine treatment of the cloned gene on a plasmid expression vector. Assaying the activity of the tailspike protein produced from this plasmid and screening for plasmid mutants were accomplished by the in situ complementation of P22 capsids imbedded in soft agar to produce infectious phage. Deletion mutations in the cloned gene have been constructed by a two step procedure involving oligonucleotide linker insertion and invitro deletion by restriction endonuclease digestion. The deletions, whose physical endpoints were determined by DNA sequencing, define 12 genetic and physical intervals into which the new mutations were mapped by marker rescue experiments. These deletions were transferred to phage P22 by recombination and used to mapmutations carried on plasmids. Following mapping, the nucleotide change for each of the mutations was determined by DNA sequencing. The majority were absolute missense mutations although both amber and ochrenonsense mutations were also identified in the protein coding portion of the gene. The suppression pattern of the nonsense mutations was determined on several nonsense suppressors. Four of the mutations cause severely depressed levels of tailspike protein expression from both the cloned gene on the plasmid expression vector and from P22 phage carrying these mutations. These mutations were identified as nucleotide changes in what is probably the P22 late operon transcription terminator which immediately follows the tailspike protein coding sequence. combined biochemical and genetic approach is studied invitro under a wide range of conditions A providing valuable in the detailed structural and (ISRAEL,ANDERSON and LEVINE1967). The factors functional analysisof a number of proteins.Prob- which control this strong, precisely timed attachment lems of protein structure and function subjected to between proteins in the capsid and tailspike protein this approachinclude DNA-protein interactions are not well understood but they should be at least (SCHMITZ,COULONDRE and MILLER1978), catalytic partially amenable to investigation by geneticap- mechanisms (KNOWLES1987), proteinstability (ALBER proaches. A clearer understanding of this assembly and WOZNIAK 1985; SCHMITZ,COULONDRE and reaction should also lead to a better understandingof MILLER1978), and protein folding (HURLE,TWEEDY less accessible assembly reactions in other supramo- and MATTHEWS 1986; JENNESS and SCHACHMAN lecular structures. The tailspike protein is also an 1983). endorhamnosidase which binds to andcleaves a-rham- The tailspike protein of Salmonella typhimurium nosyl-1,3-galactose linkages in the 0-antigen on the phage P22 has many interesting features which make surface of the host bacterium (IWASHITAand KANE- it a useful model system for this type of study. It is a GASAKI 1973). As such, it is a member of a large, well- structural component of the phage. During the last characterized group of sugar binding proteins, many step of the assembly pathway, 6 tailspike protein tri- mers attach to the phagecapsid with strong noncova- members ofwhich have known crystal structures lentbonds (KING, LENK and BOTSTEIN1973). The (QUIOCHO 1 89 6). capsid, which is the product of the penultimate step The tailspike protein is a trimeric molecule com- in P22 morphogenesis, and tailspike protein are both posed of the 666 amino acid product of P22 gene 9 easily isolated andthe assembly reaction has been (GOLDENBERG, BERGETand KING 1982; SAUERet al. 1982). Thisgene has been cloned into a plasmid ' Current address: Boyce Thompson Institute, Cornell University, Ithaca, expression vector and theDNA sequence determined New York 14853. e Current address: Department of Biological Sciences, Carnegie Mellon (SAUERet al. 1982; BERGET,POTEETE and SAUER University, Pittsburgh, Pennsylvania 15213. 1983). The protein has remarkable resistance to ther- Genetics 121: 635-649 (April, 1989) 636 J. J.and Schwarz P. B. Berget mal denaturation and to proteasedigestion. The ther- affecting a process as complex as folding of a large mal stability of the mature protein is in marked con- multisubunit protein would produce only a tempera- trast to its thermal lability during folding and subunit ture sensitive phenotype or all be located in one part assembly. The mature protein experiences only a 10- of the protein. For these reasons a genetic analysis 20% decrease in enzymatic activity after a 5-min in employing absolute lethal tailspike proteinmutants vitro incubation at 80" (IWASHITAand KANEGASAKI isolated both from the phage (BERGET and CHIDAM- 1976), whereas the amount of polypeptide forming BARAM 1989), and fromthe plasmid clone of the stable trimers in vivo drops from 90% at 27" to only tailspike proteingene has been undertaken.This 15% at 42" (GOLDENBERG,BERGET and KING 1982). analysis is possible because of the ability to comple- In vivo folding and subunit assembly of this protein ment absolute lethal phage tailspike protein mutants are coupled reactions in which the polypeptide chains with purified tailspike protein in vitro. This allows one assemble into a protrimer before maturing into the to propagate absolute lethal mutants by supplement- final trimerform. These two trimerforms can be ing either solid or liquid media with purified tailspike distinguished by theirdifferent mobilities on poly- protein. acrylamide gels and by their differential sensitivities This paper describes the construction of a plasmid- to trypsin digestion and to denaturation by 1% SDS based genetic system to facilitate the fine structure at room temperature. The mature trimer is resistant genetic analysis of this protein and its use to isolate to trypsin digestion and to SDS denaturation while and determine the nucleotide changes of 27 tailspike the protrimer is sensitive to both (GOLDENBERGand protein mutants. This system relies on a simple plate KING 1982). complementation assay to isolate mutations in the An extensive collection of absolute lethal and con- plasmid cloned version of gene 9. These mutations ditional mutations have been isolated in gene 9 have been located to 12 deletionintervals using phage (SMITH,BERGET and KING 1980;FANE and KING deletion mutants. The precise nucleotide change has 1987; BERGETand CHIDAMBARAM1989). Because been determined by DNA sequencing using a set of gene 9 is an essential gene, theoriginal genetic analysis 10 oligonucleotide primers whose complementary se- of this protein utilized conditional mutants. The most quences are spaced at approximately 200 base inter- thoroughly studied of these are the temperature-sen- vals throughoutgene 9. Significantly, the absolute sitive mutants. These mutants have nearly normal lethal missense mutations which also prevent stable thermal stabilities when they matureat permissive trimer formation u. J. SCHWARZ andP. B. BERGET, temperature but fail to form trimerswhen maturation unpublished data) are preferentially located in the is carried outat thenonpermissive temperature. They carboxy-terminal region of the proteinwhich is devoid are therefore defective in protein folding (GOLDEN- of mutations causing the temperature-sensitive phe- notype. BERG, SMITHand KING 1983). The entire set of tem- perature sensitive mutants has recently been se- quenced and the mutations are all located within the MATERIALS AND METHODS middle third of the protein's primary structure be- Phage strains and procedures: The phage strains used tween residues 141 and 493 (VILLAFANEand KING, and created in this study are shown in Table 1. General 1988). Many amber mutants also have a temperature phageP22 procedures have beendescribed (SUSSKIND, sensitive phenotype when grown on at least one sup- WRIGHT and BOTSTEIN197 1; BOTSTEIN,CHAN and WAD DELL 1972). Lambda plates and soft agar are described in pressing strain. These areapparently also defective in SIGNERand WEIL (1968). Phage strains that cannot make folding at the non-permissive temperature and they functional tailspike protein (tailspike dependent)were prop- have been mapped genetically into the same region of agated inliquid culture by addition of purified tailspike the protein as the other temperaturesensitive mutants protein at 10'' phage equivalents/ml and on plates by ad- (FANE andKING 1987). Although the analysis of mu- dition of 10" phage equivalents of tailspike protein to the soft agar overlay. Phageequivalents of tailspike protein was tants with a conditional lethal phenotype has been determined as previously described (ISRAEL,ANDERSON and valuable, especially for the investigation of protein LEVINE1967; BERGETand POTEETE 1980). folding, they represent a limited class of mutations in P22 capsids (9- particles) were made either by induction which certain expected types of mutants such as those of a gene 9 deletion mutant prophage or by a single cycle of infection by a gene 9 deletion

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