Copyright 0 1989 by the Society of America

The Isolation and Sequenceof Missense and Nonsense in the Cloned Bacteriophage P22 Tailspike Protein

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 anumber 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 aconditional 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 mutant in liquid culture. which affect attachment to the phagecapsid or endor- Induction was performed by growing a DB7000 lysogen of hamnosidase activity have been absent or underrep- P22 9- Dl 1 to 2 X 1O8 cells/ml in LB broth (LEVINE1957) resented. In fact only one conditional mutant defec- and then adding mitomycin C to a final concentration of tive in any property aside fromfolding has been 0.2 pg/ml. After overnight incubationat 37" with aeration, isolated. This is an amber mutant which produces a the cells were lysed with CHCls andthe cell debris removed by centrifugation at 10,000 X g for 10min. The capsids endorhamnosidase defective protein when grow on a were concentrated by centrifugation at 19,000 X g for 90 particularsuppressor strain (BERGET and POTEETE min and then resuspended in M9 salts (BERGETand POTEETE 1980).In addition it is unlikely that all mutations 1980) at 1/10 the original volume. Capsids were also made P22 Tailspike Protein 637

TABLE 1 Phage P22 strains

Strain Genotype Reference

P22 Ap35 gene 9::Tnl(Ap35), sieA44, TR- WEINSTOCK(1 977) P22 Ap68tdpfrlO8 gene 9::Tnl(Ap68), sieA44, TR+ YOUDERIANand SUSSKIND(1 980) P22 Dl P22 9A(-38 to 69), sieA44 This study P22 D2 P229A(72 to 427), sieA44 This study P22 D3 P22 9A(229 to 510), sieA44 This study P22 D4 P22 9A(430 to 601), sieA44 This study P22 D5 P22 9A(513 to 724), sieA44 This study P22 D6 P22 9A(604 to 979), sieA44 This study P22 D7 P22 9A(7 13 to 999), sieA44 This study P22 D8 P22 9A(982 to 1513), sieA44 This study P22 D9 P22 9A(998 to 1434), sieA44 This study P22 Dl0 P22 9A(1422 to 1513),sieA44 This study P22 Dl 1 P2219 9A( 15 to 1826),sieA44 This study P22 Dl 2 P22 9A(1752 to 2058), sieA44 This study P22 hmH5 P22 9- (Q59 amber), sieA44 This study P22 hmH19 P22 9- (W640 amber), sieA44 This study P22 hmH7, hmH30-3-3P22 (G to A at 201 7), sieA44 This study P22 hmH3-5 P22 P22 (G to A at 2019), sieA44 This study P22 hmH3O-1-1 P22 (C to T at 2029), sieA44 This study by infecting DB7000 with P22 Ap68tdpf1-108at a multiplic- stant between colonies, the size of the halo is a crude ity of 5 phage per cell. After 90 min of incubation with indicator of the quantity or specific activity of tailspike aeration the cells were lysed with a few drops of CHC13. protein produced. Removal of cell debris and concentration of the capsids were General recombinantDNA techniques: Small scale plas- by the same procedure as for the induced prophage. Induc- mid preparations were made by the procedureof BIRNBOIM tion of a prophage produces around 2 X 10' capsids from and DOLY(1 979). Largescale purification of plasmid DNA 40 ml of culture, whereas infection produces around 5 X was by the CsCl/ethidium bromide density gradient method 10" capsids from 40 ml of culture. (DAVIS,BOTSTEIN and ROTH 1980). Purification of DNA Lysates of P22Ap35, which has the transposon Tnl from low melting point agarose gels was by the method in inserted into gene 9, were prepared by inducing the pro- MANIATIS,FRITSCH and SAMBROOK(1982). TE is 10 mM phage in the mannerdescribed above. Tailspike protein was Tris and1 mM EDTA, pH 8.0. Restriction enzymes, Klenow attached to the phage capsid by adding tailspike protein to fragment of DNA polymerase I, S1 nuclease and T4ligase the phage lysate to a concentration of 10" phage equiva- were purchased from New England Biolabs, BRL Inc., and lents/ml and incubating at 37" for atleast 2 hr. Thephage Boehringer Mannheim and used according to the manufac- were then concentrated as described above. DNA was iso- turers' recommendations. lated from P22 by the method used for phage X (MANIATIS, Construction of pJS27 and pJS28: The tailspike protein FRITSCHand SAMBROOK1982). gene,gene 9, was originally cloned into pPB13 and ex- Bacterialstrains and procedures: Bacterial stocks for pressed from the lacUV5 promoter (BERGET,POTEETE and plating P22 were made by growing DB7000 in LB broth to SAUER1983). This plasmid contains the origin of plasmid approximately 2 x 10' cells/ml, pelleting by centrifugation, replication and the ampicillin resistance gene of pBR322 and resuspending them inLB broth at 1/10 the original and approximately 4 kb of P22 DNA. The plasmid pJS27 volume. Competent cells for transformation were prepared was derived from this plasmid through removal of roughly by the CaC& method (DAVIS,BOTSTEIN and ROTH 1980). 2 kb of P22 DNA from an AluI site near the 3'-end of gene The strain Escherichia coli RR1 was the recipient strain for 9 to the PuuII site which is the junctionof pBR322 and P22 most of the recombinant DNA work (Table 2). DNA (Figure 1). There are several AluI sites in pPBl3, so Plate complementation assayof tailspike protein activ- this deletion was made by performing a partial digest with ity: Colonies of bacteria containing plasmids which express AluI in the presence of ethidium bromide(PARKER, WATSON gene 9 are assayed for functional tailspike protein by trans- and VINOGRAD1977) followed by a complete digestion with ferring them with toothpicks to a lambda agar plate that has PvuII. A linear DNA fragment of the expected sizewas 2.5 ml of soft agar overlay containing 10' P22 capsids and purified from a low melting point agarose gel and the ends 2 X 10' DB7000 cells. The plates are then incubated over- ligated together. The resulting plasmid, pJS27,has only night at 30". Tailspike protein is supplied to the embedded about 350 bp of P22 DNA between the putative transcrip- capsids by the transferred colonies through release into the tion terminator of gene 9 and the beginning of pBR322 surrounding agar, presumably through cell death. When DNA. The plasmid pJS28 was derived from pJS27 by incor- functional tailspike protein attachesto P22 heads they infect porating the M 13 origin of single stranded replication and and lyse the surroundingbacteria to form a large plaque or DNA packaging from pZ152 (ZAGURSKYand BERMAN halo around thecolony being tested. The efficiency at which 1984). This was done by replacing DNA from the NdeI site this infectious process occurs depends on the amount of to theEcoR1 site of pJS27 with the corresponding fragment tailspike protein and its functional activity. If the amountof of DNA from pZ152 which contains the M13 origin. The cells transferred to the indicator plate is kept roughly con- M13 origin makes it possible to isolate single stranded 638 J. J. Schwarz and P. B. Berget

TABLE 2

Bacterial strains

Strain Genotype Reference/Source E. coli Bb Prototroph E. coli GE2343 F’A(lac, pro)XIII, ara,argE G. WEINSTOCK am, gyrA, rpoB, met B, supB E. coli GF.2344 F’A(lac, pro)XIII, ara, argE G. WEINSTOCK am, gyrA, rpoB, met B, supC E. coli JM 103 A(1ac-pro), thi, strA, supE, MESSING and VIEIRA (1 982) endA, sbcB, hsdR, F’traD 36, proA, proB, lacP, lacZ AM15 E. coli KK2 186 Same as JM103 except P1- ZACURSKYand BERMAN(1 984) E. coli MC1061 araD 139, A(ara, leu)7697, CASADABAN and COHEN(1980) Alac X74, strA, galU, galK, hsr E. coli RRl F-, hsd S20 (rB-, mB-), aral4, BOLIVARet al. (1 977) proA 2, lacy 1, galK 2, rpsL 20 (Smr), xyl-5, mtl-I, supE 44, 1- S. typhimurium DB7000 leuA am4 14 SUSSKIND,BOTSTEIN and WRIGHT (1974) S. typhimurium DB7004 leuA am4 14,supE SUSSKIND,BOTSTEIN and WRIGHT(1 974) S. typhimurium DB7 154 leuA am414, hisC am527, WINSTON,BOTSTEIN and MILLER(1 979) supD 10 S. typhimurium DB7 156 leuA am414, hisC am527, WINSTON,BOTSTEIN and MILLER(1979) supF3O S. typhimurium DB7 157 leuA am414, hisC am527, WINSTON,BOTSTEIN and MILLER (1 979) supJ60 S. typhimurium MS 1868 leuA am414, r-, m+, felss RENNELL andPOTEETE (1985) plasmid DNA for sequencing and oligonucleotide mutagen- by treatment with DNase I to make a single-stranded break esis by infecting with a filamentous helper phage and puri- in the phosphodiester backbone of the plasmid (GREEN- fying the DNA from phage capsids (ZAGURSKYand BERMAN FIELD,SIMPSON and KAPLAN1975) andlinearizing the plas- 1984). mid at this break with S1 nuclease. A linker was also inserted Construction of gene 9 deletions in plasmids: Twelve into either the unique NcoI or BamHI restriction site in gene 9 deletions were made for use in deletion mapping. gene 9 by digesting the plasmid with the appropriate en- These deletions were made inplasmids and then moved zyme, filling in single stranded ends with the Klenow frag- onto phage. The plasmid deletions were made by essentially ment of DNA polymerase I, and ligation in the presence of the same two-step procedure as used by SHORTLE(1 983) for theHindIII linker. A Hind111 site was created in P22 construction of deletions in the staphylococcal nuclease sequences 5‘ to the startof gene 9 at position -39 from the gene. In the first step an 8-base HindIII linker, 5’ first codon of the mature protein by oligonucleotide di- CAAGCTTG 3‘ (Collaborative Research, Inc.) was inserted rected mutagenesis using the oligonucleotide 5’ into gene9 at various positions to make plasmids containing CCGTAGCCAAGCTTCGGCAATTCC 3‘. Thisand all a single HindIII site. Insertion of the linker into gene 9 other oligonucleotides were made on an Applied Biosystems should in general inactivate gene 9. Therefore plasmids with Model 380A DNA synthesizer unless specifically indicated HindIII linker insertions in gene 9 were identified as trans- and were purified by the procedure recommended by the formants which had lost tailspike protein activity in the plate manufacturer. The mutagenesis was done by the method of assay. The HindIII insert into the AluI site beginning at ECKSTEIN(TAYLOR, OTT and ECKSTEIN1985) with reagents nucleotide 205 1 was isolated in the screen for transformants and procedures supplied by Amersham. The locations of which had lost tailspike protein activity. This insertion is the unique HindIII sites in all gene 9 containing plasmids outside of gene 9 and in subsequent plate assays was found are shown in Figure 2A. to not inactivate the gene. Oncea sufficiently large set of plasmids containing The location of the HindIII linker insertion depends on HindIII sites in gene 9 was produced, they were used to the method used to linearize the plasmid. A number of make deletions in the second step of this procedure which methods for linearizing the plasmid were used in order to is outlined in Figure 2B. The basic scheme for construction obtain insertions at useful positions throughout the gene. of each deletion was to digest two plasmids with HindIII Most insertions were made semirandomly by partial diges- linker insertions in different locations with HindIII and tion of plasmid DNA and either AluI, HaeII, or Hue111 in another enzyme that cuts only once in the plasmid, purify the presence of ethidium bromide (PARKER,WATSON and the fragments containing the 5’ and 3‘ junctions of the VINOGRAD1977) followed by purification of full length desired deletion from a low melting point agarose gel, and linearized plasmids from low melting point agarose gels. ligate the fragments together. The resulting plasmid is miss- The linear plasmid molecules were then circularized in the ing the DNA between the two HindIII linkers and retains a presence of the linker with T4 DNA ligase. Two insertions Hind111 site marking the endpoints of the deletion. The were made by cleavage of the plasmid at random positions deletion D9 was made by inserting aHindIII linker into the P22 Tailspike Protein 639

Eco R1 A

nGene9 I I I I I 1 500 1000 1500 2000

B Eco R1

Eco R1 Ecp R1

Eco+ R1 EdR1 baGaegHin dm / Nde I .-%:oNdeI

Eco R1

N& I FIGURE2.-Construction of plasmid deletions. A, Location of HindIII sites created in gene 9 are indicated by vertical lines above FIGURE 1.-Construction of pJS27 and pJS28. The plasmid the gene. These include the location of theHindIl1 linker insertions pJS27 was derived from pPBl3 by partial digestion with Ah1 into both pPBI3 and pJS27; and the HindIII site in pJS28 created followed by digestion to completion with PvuII, and ligation. The by oligonucleotide mutagenesis. B, Gene 9 deletions were con- plasmid pJS28 was constructed by digestion of pJS27 and pZ152 structed by digesting two plasmids with HindIII sites at different with EcoRI and NdeI, mixing the digested plasmids together, and locations in gene 9 with Hind111 and anotherenzyme that cuts only ligation. The MI3 origin of DNA replication and single strand once in these plasmids, usually EcoRI. The fragments comprising packaging is indicated by the anti-clockwise arrow on pZ152 and the 3' and 5'junctions of the deletion were isolated from agarose pJS28. The arrow indicates the 5' + 3' polarity of the packaged gels and ligated together. single strand. The smaller arrowhead indicates the location of the lacUV5 promoter found in plasmids pPB13, pJS27 and pJS28. P22 Ap35 at a multiplicity of infection of 10. The infection BamHI site in gene 9 of a plasmid which already carried a was allowed to continue for 2 hr at30" after which the cells HindIII linker to make a plasmid with two HindIII sites. were lysed with CHCls andthe cell debris pelleted by The region between the HindIII sites was then deleted by centrifugation in a microfuge. The lysate was plated on digesting with HindIII and ligating under dilute conditions DB7000 with tailspike protein. The plaques that arose were which favor intramolecular ligation. The deletion Dl 1 was replica plated using toothpicks onto a plate with a lawn of made by digesting plasmid DNA with BamHI and HpaI, DB7000 and a similar plate which also contained tailspike protein to identify those which were tailspike dependent. filling in the single stranded BamHI ends with Klenow The tailspike dependent phage were presumed to carry the fragment of polymerase I, and ligating the plasmid under gene 9 deletion and were further tested. The endpoints of dilute conditions in the presence of HindIII linkers. deletions Dl and D2 had been sequenced prior to being Transfer of deletions from plasmidsto phage:Deletions moved to phage, so the transfer of these deletions to phage were transferred from plasmids to phage by recombination was confirmed by digesting the phage DNA with HindIII between P22 Ap35and the desired plasmid. P22 Ap35 and observing the presence of new restriction fragments of carriesa Tnl element in gene 9 and thuscannot form the expected size. The otherplasmid deletion endpoints had plaques even in the presence of tailspike protein because the not been sequenced prior to transferring to phage so they phage genome is oversized and thus lacks terminal redun- were sequenced after moving them to the plasmid pJS28 as dancy when packaged into particles. Replacement of the described below. Tnl element with the deletion by in vivo recombination Transfer of deletions from phageto the plasmid pJS28: restores terminal redundancy and allows the phage to form P22gene 9 deletions were transferredto pJS28 during plaques on plates with tailspike protein. S. typhimurium specialized transduction of this plasmid by P22. The trans- MS1868 containing a plasmid with a gene 9 deletion was ducing lysate was prepared by infecting mid-log phase grown to approximately 2 X 10' cells/ml and infected with DB7000 (pJS28) with aP22 gene 9 deletion phage at a 640 J. J. Schwarz and P. B. Berget multiplicity of 5 phage per cell. The transduction was done without the addition of tailspike protein to detect tailspike by incubating 0.2 ml of DB7000 plating bacteria with 0.1 independent recombinant phage. ml of the lo-’ and 10-4 dilutions of the transducing lysate In vitro heteroduplex deletion mapping: The for 10 min at 37”. This mixture was then spread onto the hmH 10 has a dominant negative phenotype, so it is difficult selection plates. The cells which received the plasmid were to map using deletions carried on phage because mutant selected for on green indicator plates (CHENet al. 1972) protein produced in large quantities from the plasmid can containing 150 rg/ml ampicillin and 10 mM EGTA (WEIN- attach to the wild-type recombinant phage to make them STOCK, SUSSKINDand BOTSTEIN1979) andincubated at 37 ” . noninfectious. We therefore mapped this mutation by the Green indicator plates contain (per liter) 8 g tryptone, 1 g heteroduplex mapping protocol of SHORTLE(1 983) in which yeast extract, 5g NaCI, 15 g agar,and 21 ml of 40% aheteroduplex is formed between a single strand from glucose, 25 ml of 2.5% (w/v) Alizarin Yellow G, GG plasmid containing the mutation and a single strand from a (MATHESON, COLEMANand BELL)and 3.3 ml of 2% (w/v) plasmid containing a deletion. This heteroduplex is made water-soluble Aniline Blue (BDH Chemicals). Stably lyso- in vitro by denaturing a mixture of the two plasmid mole- genized cells produce large light green colonies on such cules linearized at different restriction enzyme sites, allow- plates while unstable lysogens or phage-infected colonies ing the linear molecules to renature to form both parental form small dark green colonies (SMITHand LEVINE 1967). linear molecules and circular heteroduplexes. This plasmid The ampicillin-resistant colonies were pooled and used to DNA mixture is transformed into competent cells where the make a rapid plasmid DNA preparation. This plasmid DNA heteroduplexes are resolved in vivo. The linear molecules was used to transform E. coli MC 1061 or KK2 186 toampi- transform with such a low frequency that generally only cillin resistance. These colonies were thenscreened for circular heteroduplexes are successfully introduced into the tailspike protein productionin the plate assay. Plasmidsfrom recipient cells.If the mutation is not within the deleted cells that did not make functional tailspike protein were region then both mutant and wild-type plasmids will result presumed to carry the gene9 deletion. This was confirmed from the heteroduplexdepending on thestrand choice by demonstrating the presence of a Hind111 site in these during mismatch repair. In contrast, if the mutation is within plasmids and subsequent DNA sequencing. The extent of the deleted interval only plasmids carrying the mutation will the missing DNA inthese deletions has been determined by result. DNA sequencing. In preparation for heteroduplex mapping the mutation Hydroxylamine mutagenesisof plasmih CsCl gradient hmH10 was moved from pPBl3 onwhich it was isolated to purified plasmid DNA was mutagenized by a modification pJS28 in an EcoRI to HpaI fragment which contains about of the procedure described by HUMPHREYSet al. (1976). 90% of the tailspike protein gene. This was done by digest- The only difference from that procedurewas the DNA was ing pPBl3 hmHlO andpJS28 with EcoRI and HpaI,isolat- incubated in the hydroxylamine solution for 30 min at 65” ing the fragments from alow melting point agarose gel, and followed by dialysis against two changes of 1000-fold excess ligating the fragments with T4 ligase. The recombinant of TE before transformation into E. coli Bb, MC106 1, or plasmid was tested for tailspike protein activity with the JM 103. plate assay to determine if the mutation was transferred on the EcoRI to HpaI fragment. Because this plasmid failed to In vivo deletion mapping: Deletion mapping of mutant plasmids was done by two procedures-a spot test in which produce a halo in the plate test it was presumed to contain the hmH 10mutation. Heteroduplexes were made between the progeny of recombination were not isolated and a sen- this plasmid and selected gene 9 deletions carried on pJS28 sitive marker rescue test in which the progeny of the crosses by the procedureof SHORTLE(1 983). After transformation were isolated and tested. The tailspike dependent phage of the heteroduplex reactions into MC1061 the ampicillin which were used in these mapping procedures had tailspike resistant colonies were assayed for functional tailspike pro- protein added to them exogenously so that they could infect tein (wild-type recombinants) with the plate assay. cells once. The spot test was done by transferring approxi- DNA sequencing: DNA sequencing was done by the mately lo6 deletion phage in 10 rl onto a plate containing dideoxy method(SANGER, NICKLEN and COULSON 1977) S. typhimurium MS1868 carrying the mutant plasmid in the using the following 10 oligonucleotide primers: P1 (5‘ soft agar. All of the different phage deletion strains were GCGGCAATTCCTTGC 3’) P2 (5’ ATCAACGCA- transferred atone time from amicrotiter dish using a GCCGGTA 3’), P3 (5’ ATTGCAGGATGCAGC 3‘), P4 multiple prong applicator from West Coast Scientific. The (5’ CAAGCCTTGGACGGA 3’), P5 (5’ AATGTA- plates were then incubated overnight at 30”. Because the TAGGGGTCG 37, P6 (5’ CAGTAAGTAGCGCCCA 3’), phage deletion strains do not revert to tailspike independ- P7 (5’ GCTGACACTGACATGA 3’), P8 (5’ ACCTACT- ence at a detectable rate, positive marker rescue was scored CACCCACGA 3’), P9 (5’ GGTAGACCCCTCTAGA 3’), by the presence of any plaques in the spots. Mapping ambi- P10 (5’ GCACACCTGACGCTG 3’). These oligonucleo- guities caused by failure to marker rescue with nonoverlap- tides are complementary to gene 9 DNA located at approx- ping deletion intervals were usually resolved by spotting a imately 200 base intervals along the gene. DNA sequencing larger number of phage using a pipet. Marker rescue was from these primers is in the same direction as gene 9 only detected in the cross between Dland hmH4-1 by transcription. plating 10-fold morephage than usual. Sensitive marker Mutations were isolated intwo plasmids, pPB13 and rescue was done in liquid culture. S. typhimurium MS1868 pJS28. The plasmid pPB13 does not have the M13 origin, carrying the mutant plasmid was grown to mid-log phase so before mutations in this plasmid were sequenced they and infected with phage carrying each of the gene 9 dele- were either subcloned into pJS28 or the M13 origin was tions at a multiplicity of 5 phage percell. To ensure thatall inserted into theplasmid carrying the mutation. The choice of the progeny were infectious, tailspike protein was added of procedure depended on thelocation of the mutation with to the culture to a final concentration of 5 X 10” phage respect to restriction enzyme sites that could be used for equivalents/ml. After a 90-min incubation at 30” the cells subcloning. Single stranded plasmid DNA was produced by were lysed with CHCI, and thecell debris removed by a 10- infection of E. coli KK2186 containing the mutant plasmid min centrifugation in a microfuge. The lysate was diluted with phage IR1 according to the method of DENTE,CB- and 0.1 ml of a 100-fold dilution was plated on DB7000 ARENI and CORTESE(1983). DNA sequencing was done P22 Tailspike Protein 64 1 Gene 9 Thr-1 Leu-666 Dl Ala-24 D2 LyS-23 D2 - Gly-143 D3 -Asn-75 Ala-171 D4 Asp-142 pro-201 D5 Lye170 Phe-242 D6 Ser-327 Thr-200 D7 Lys-241 Ala-334 D8 Gly-505 Thr-326 D9 ser-333 Asp479 FIGURE3.-Deletion map of gene Phe-472 Gly-505 9. The bars in this diagram represent Dl 0 the extent of gene Y DNA present in

Dl 1 Ile-506 7HiS-601 each of the deletions reported in this Gly-582 paper drawn to scale. In addition, the Dl2 deletions previously reported (BERGET pMC2 -Ala-90 andCHIDAMBARAM 1989) are indi- PMG La-214 cated for comparison. The first line LyS-225 represents the entire gene Y sequence. pMC26 The complete codons which are near- pMC26 ne-245 est the endpoints of the deletions are pMC6 lle-387 indicated above each bar. The dele- tions Dl through Dl 2 exist on both pMC12 Asp399 plasmids and phage (Table 1). pMC9 pMC13 pMCl0 pMCl5 pMC16 pMCl8 pMC25 Lvs-631 pMC21 Lvs-651 pMC23

using rea ents and procedures provided by BRL, Inc. The cisely located by sequencing across the deletion end- 32Pand kS dATP were supplied by New England Nuclear. points in the plasmid version of each deletion. The deletion D9 has 160 bpof pBR322 DNA inserted into RESULTS gene 9 between the HindIII linker and the 3‘ region of gene 9 DNA. The inserted DNA is composed of 2 Gene 9 deletions: HindIII octomerlinker insertions segments of pBR322: the segment nearest the 5’ end intogene 9 and flanking sequences were made in of gene 9 is composed of DNA from position 2580 to plasmids pPB13 and pJS27 and pJS28 as described in 2550 and the 3’ segment is from 3713 to 3841 in MATERIALS AND METHODS. Recombinant DNA tech- niques were used to combine fragments of these plas- pBR322 (SUTCLIFFE1979). The arrangement is such mids to generate deletions in gene 9 and flanking that pJS28 Dl0 contains an inverted repeat of the 5’ sequences whose endpoints are marked by a HindIII segment of pBR322 DNA and a direct repeat of the site in the resulting plasmids. The location and extent 3’ segment. This insertion of DNA occurred when of these deletions (Dl through Dl2) is represented in the HindIIIlinker insertion that formsthe 3’junction Figure 3 along with the deletions previously reported of Dl 0 was constructed. The plasmid was linearized (BERGETand CHIDAMBARAM1989). Each of the dele- by successive treatment with DNAse I and S1 nuclease tions was crossed onto P22phage; and those deletions which evidently created many small DNA fragments which did not originate on the plasmid pJS28 were that were ligated into the linearized plasmid along subsequently crossed fromthe resulting deletion with the HindIIIlinker. This insertion went unnoticed phage onto pJS28 in preparation for sequencing as until the deletion junction was sequenced. Some of described in MATERIALS AND METHODS. Each P22 thesurrounding DNA sequence andthe HindIII phage which carries a deletion in gene 9 along with linker which marks the position of the deletion is its genotype given as theextent of gene 9 DNA shown for each deletion in Table 3. deleted is listed in Table 1. The deletions were pre- Isolation of gene 9 mutations on plasmids: pPB 1 3 642 J. J. Schwarz and P. B. Berget

TABLE 3 Deletion mapping of mutants: The mutant plas- Sequence of deletion junctions" mids were moved by transformation from the E. coli

______strains in which they were isolated into S. typhimurium 1-39 170 MS 1868 in preparation for deletion mapping. For the Dl ...CGTAGCCAAGCTTGCTGTTGC.. . majority of the mutants the rapid spot test described 1711428 in MATERIALS AND METHODS was sufficient to locate D2 ...TTAAAGCAAGCTTGCCTTCTT ... the mutants to within a given deletion interval. 1228 1511 Seven of themutants (hmH4, hmH7, hmH3-3, D3 ...CAACGGCAAGCTTGCTAAATT. .. hmH3-5, hmH3O-1-1, hmH30-2-3 and hmH30-3-3) 14291602 could not be mapped with the spot test because cell D4 ...TGATGGCAAGCTTGCATGGGT ... lysis occurred in crosses with phage carrying each of 15121725 the deletions. Because four of these mutants (hmH7, D5 ...GTAAAGCAAGCTTGTCCCAGG ... hmH3-5,hmH30-1-1 and hmH30-3-3)complement 16031980 phage capsids in the plate complementation assay by D6 ...ACACCACAAGCTTGCTAT GGG... making small halos as described above, it was thought 171211000 that weak complementation was occurring in at least D7 ...TAAGCGCAAGCTTGGCCCAGT. .. the case of these four mutants to obscure the marker 1981 11514 rescue results. Therefore sensitive marker rescue ex- D8 ...AACCAGCAAGCTTGGATCCGC. .. periments were done to determine the phenotype of 199711435 the progeny under conditions where complementa- D9 ...TAAGTACAAGCTTG***GACAC ...* tion is not observed.Sensitive marker rescue was done 11421 11514 Dl0 ...TACCAACAAGCTTGGATCCGC ... by crossing these seven mutants in liquid culture with phage carrying deletions and testing the progeny for I1518 11827 Dl 1 ...TGGGATCAAGCTTGCCACAAA.. . their ability to grow on plates containing S. typhimu- rium but no tailspike protein. Two of the mutants, 11751 12059 Dl 2 ...GGGGGGCAAGCTTGCTAATTA ... hmH3-3and hmH30-2-3, produced phage which were tailspike-independent for plaque formation a The numbering system is such that number 1 corresponds to the first nucleotide of the first codon of the N-terminal amino acid when crossed withall deletions but Dl 1 where no (Thr) in the mature protein. The numbers above each sequence tailspike-independent plaques were produced. These refer to the last 3' nucleotide and the first 5' nucleotide of the sequence which is identical to the phage P22 sequence flanking the plasmid mutations must have produced tailspike pro- deletion. The Hind111 linker sequence is underlined. tein which could weakly complement deletion phage ' (***) indicates an unexpected insertion of 160 bases of pBR322 in the spot test but were unable to produce enough DNA (see text for description). tobe detected in the plate assay. Fourmutations and pJS28 plasmid DNA was mutagenized with hy- (hmH7, hmH3-5, hmH30-1-1 and hmH30-3-3) pro- droxylamine and then transformed into three differ- duce progeny phage which make very small plaques ent E. coli strains (Bb, JM103 and MC1061) as de- when crossed with the deletion Dl 2. When crossed scribed in MATERIALS AND METHODS. The purpose of with all other deletions the progeny phage produce a trying different strains was to find the strain in which mixture ofvery small and normal size plaques. So tailspike protein mutants with low amounts of activity these mutants have a small plaque phenotype when could most easily be distinguished from wild type in they are on phage and are located within the region the plate assay. The strain MC 106 1 provedto be the of Dl 2 that does not overlap Dl 1. All of the small most reliable indicator of tailspike protein activity plaque phenotype mutants, hmH7, hmH3-5, hmH30- simply because this strainproduces the largest size 1-1 and hmH30-3-3 are able to complement phage halo with the wild-type gene. This is probably due to capsids extracellularly in the plate assay to make small the fact that the lac repressor gene is deleted in this halos. Crosses between all of the deletions and the strain so expression of protein from the plasmid lac mutant hmH4 produced plaques of normal size. Be- UV5 promoter may be higher than in strains which cause the mutations which make small halos when on contain the lac repressor. The transformants were plasmids also make small plaques when on P22, it is screened by the plate assay for halo production as unlikely that a mutant such as hmH4 which does not described in MATERIALS AND METHODS. Twenty-seven make a halo would make a normal size plaque when independent mutants were isolated froma total of crossed onto P22. Therefore the most likely location 13 10 colonies screened. Four of these, hmH7, hmH3- of a plasmid mutation with a phenotypesuch as hmH4 5, hmH3O-1-1 and hmH30-3-3 generate smaller than is in the lac UV5 promoter region rather thanin P22 normal size halos compared to wild type while the rest DNA. DNA sequencing later showed that hmH4 is a reproducibly generate no halos at all. C + T change at -32 in the promoter which is the P22 Tailspike Protein 643 same site as the C Apromoter down mutation GE2343 and GE2344 which insert Gln and Tyr, re- L157 (REZNIKOFFand ABELSON1978). spectively, at the ochre codon. The tailspike protein It was difficult to map conclusively the mutation activity was tested by the plate assay. The results for hmHlO using the phagedeletions because the fre- ochre suppression were fairly uniform (Table 6). Al- quency of wild type phage produced in both the spot though the halo size was small, there was no distin- test and the sensitive marker rescue test was very low. guishable difference between the wild-type glutamine As will be more fully discussed in a later paper, the and the tyrosine substitution at codons 11 2, 226 and mutation hmH 10 produces a tailspike protein which 592. The nonpermissive temperaturefor tempera- attaches to phage capsids but is defective in the en- ture-sensitive tailspike protein mutants is 39". How- dorhamnosidase activity which is required for infec- ever, the halo size of suppressed ochre mutants was tion. It islikely thatthe protein produced by the almost imperceptibly small at39", even when the mutant hmH10, which is overproduced from theplas- correct amino acid was being inserted, so it was not mid, attaches to the progeny phage capsids generated possible to compare the effect of different amino acid in the mapping crosses to make them noninfectious insertions at thenonpermissive temperature. The for- regardless of theirgenotype (a dominant negative tuitous occurrence of a proline to serine change at phenotype). This mutation was therefore mapped us- codon 1 10 in combination with the ochre mutation at ing the plasmid-based heteroduplex deletion mapping codon 112 in hmH30-2-4 made it possible to test the procedure of SHORTLE(1983) as described in MATE- effect of Pro 1 OSer1 both by itself and in combination RIALS AND METHODS. The recombination products of with Glnl 12Tyr. Neither of the changes affects the this mapping protocol are clonally isolated plasmids protein to an extent thatcan be detectedby this assay. residing in individual colonies. These plasmids are Because there is an extensive set of amber suppressors assayed independently for production of functional in S. typhimurium and thelevel ofsuppression of amber tailspike protein in the plate assay. Therefore there mutations on phage is easier to determine than on should be no competition between mutant and wild plasmids, hmH5 and hmH19 were crossed onto P22 type proteinsfor phage capsids to complicate the and tested on these suppressor strains. The transfer interpretation of the mapping results. The results of theamber mutations to P22 was confirmed by from the heteroduplex mapping of hmH10 indicate crosses between the presumedP22 ambermutants that hmH 10maps to deletion intervals D8 and D 10. and the identical allele on the plasmid, as well as with With the exception of hmH4, each of the mutations nearby hmH mutations. Backcrosses to plasmids car- isolated on the cloned gene9 could be unambiguously rying the identical allele failed to produce tailspike located genetically in one of the deletion intervals independent phage above the background reversion described above. The results of the deletion mapping level, whereas recombination toproduce wild type experiments aresummarized in Table 4. was detected for plasmids with nearby mutations. In DNA sequence of hmH mutants: Once the muta- addition, because hmH5 creates a new XbaI site, its tions had been located within a deletion interval of transfer was confirmed by restriction enzyme diges- known physical size, the exact nucleotide change for tion of isolated phage DNA. The phage carrying the each mutation was determined by DNA sequencing amber mutations were then plated on the different using the appropriate primers describedin MATERIALS suppressor strains and incubated at either 30" or 39" AND METHODS.For each mutation the entire interval to assess the effect of different amino acid substitu- inwhich the mutation was genetically located was tions. The results for amber suppression are more sequenced to insure that every nucleotidechange diverse than for ochre suppression. The requirement within that region was found. The nucleotide and for Trp at codon 640 is stringent because four other inferred amino acid changes of all the mutants are amino acids at this site failed to produce infectious listed in Table 5. All of the changes are the expected phage particles above the levelof reversion of the outcome of the deamination of cytosine by hydroxyl- amber mutation (Table 6). In contrast, insertion of amine. Even thoughthe mutations hmH15 and the amino acids Ser, Leu and Tyr at codon 59 pro- hmH30-2-5,and hmH7 and hmH30-3-3 have the duced functional infectious phage at about the same same nucleotidechanges they are independent iso- level as insertion of the wild-type amino acid, Gln, at lates. both 30" and 39". Suppression of nonsense mutations: Several of the Mutations in the transcription terminator:Muta- hmH mutants contain nonsense mutations. Suppres- tions in the plasmid copy of gene 9 which caused the sion of these mutations by different suppressor strains production of small halos in the plate assay were provides information on the suitability of different intentionally sought for the purpose of finding mu- amino acids at a particular position. The ochre mu- tants which make a tailspike protein with decreased tations were suppressed by moving the plasmids with specific activity. This type of mutant iseasily over- thesemutations into the two suppressorstrains looked in screens of mutagenized phagebecause small 644 J. J. Schwarz and P. B. Berget

TABLE 4 Deletion mapping of gene 9 mutations'

Deletion intervals

DlD3 D2 D4D6 D5 D7 D8 D9 Dl Dl0 1 Dl2 Spot testb Mutant hmH4-1 + - + + + + + + + + + + hmH5 + - + + + + + + + + + + hmH3 + - + + + + + + + + + + hmH30-3-4 + - - + + + + + + + + + hmH30-2-4 + - - + + + + + + + + + hmH 15 + - - + + + + + + + + + hmH30-2-5 + - - + + + + + + + + + hmH30-3-5 + + + + - - + + + + + + hmH30-2-1 + + + + + - - + + + + + hmH12 + + + + + + + - - + + + hmH5-16 + + + + + + + - + - + + hmH 1 + + + + + + + + + + - + hmH2 + + + + + + + + + + - - hmH30-2-2 + + + + + + + + + + + - hmH14 + + + + + + + + + + + - hmH 19 + + + + + + + + + + + - hmH30-3-6 + + + + + + + + + + + - hmH3-2 + + + + + + + + + + + - hmH30-3-7 + + + + + + + + + + + - Sensitive marker rescue' Mutant hmH4 + + + + + + + + + + + + hmH3-3 + + + + + + + + + + - + hmH30-2-3 + + + + + + + + + + - + hmH7 + + + + + + + + + + + S hmH30-3-3 + + + + + + + + + + + S hmH3-5 + + + + + + + + + + + S hmH3O-1-1 + + + + + + + + + + + S Heteroduplex deletion mappingd Mutant hmH 10 + + + + + + nd - nd - + +

a (+) indicates marker rescue, (-) indicates lack of observable marker rescue, (s) indicates small plaques were observed when the progeny of the cross were plated, and (nd)indicates the heteroduplex was not made. The spot test was done by spotting P22 gene 9 deletion phage on a lawn of S. typhimurium containing plasmids with the mutant gene 9. ' Sensitive marker rescue was done by crossing the deletion phage with the mutant plasmids in liquid culture and plating the progeny. Heteroduplexes were made between plasmids with deletions and with the hmH10 mutation, transformed into E. coli and tested for marker rescue in the plate complementation assay. plaque mutantscan also arise from mutations in other HmH mutants which createRFLPs: Missense mu- . Four small halo mutants were isolated. All of tations which cause the creation or loss of restriction these were moved to phage during the process of enzyme sites lead to restriction fragment length poly- deletion mapping by the sensitive marker rescue tech- morphisms (RFLPs). RFLPs are useful markers for nique and found to make very small plaques. Unex- the presence of alleles during thetransfer of mutations pectedly none of these mutations were identified by between Plasmids and Phage and during reversion DNA sequencing to be in the protein coding sequence analysis. Such was the case for hmH5as well as others of the gene but rather in the sequence 3' to the stop listed in 5. codon which has been suggested to be the transcrip- tion terminator of the P22 late operon (SAUERet al. DISCUSSION 1982). As shown in Figure 4, these mutationsindivid- There is currently an impressive collection of mu- ually change three of the four GC pairs in the stem of tagenic techniques applicable to genes which are the proposed stem and loop structure to eitherAC or cloned in plasmids and single stranded vectors. Some GU pairs which decrease the calculated stability of the of these techniques even permitisolation of mutations structure from the wild-type AG of -21.4 kcal/mole without aphenotypic screen (MYERS, LERMANand to -13.55 kcal/mole (hmH7, 3-5, and 30-3-3) or MANIATIS1985). Although it is possible to undertake -1 1.7 kcal/mole (hmH30.1.1) (SALSER1977). the genetic analysis of a cloned gene without a rapid Tailspike Protein P22 Tailspike 645 TABLE 5 Nucleotide and amino acid substitutionsof gene 9 mutants

A mino acid Protein acid Amino RFLP M utant Nucleotide change” changeb activity’ changeb change” Nucleotide Mutant generatedd Missense mutations hmH4-1 G-A82 G 28 R ND hmH3 G + A 209 G 70 D ND Lost HPaII hmH30-3-4 G+A 298 D 100 N ND New HpaI hmH30-2-1 G-A 860 C 287 Y ND Lost Mae111 hmH 12 C+T 1193 A 398 V Nd New HincII hmH5-16 G + A 1480 G 494 R ND New Mae1 hmH10 G-A 1513 G 505 R ND hmHl G + A 1637 G 546 D ND hmH3-3 G+A 1688 R 563 Q ST New PstI hmH30-2-3 G+A 1723 A 575 T ST hmH30-2-2 G +A 1844 G 615 E ND hmH14 G + A 1849 E 617 K ND hmH3-2 C + T 1961 P 654 L ND hmH30-3-7 C + T 1991 A 664 V ND New Mae111 Nonsense mutations hmH5 C+T 175 Q 59 Amber ND New Xbal hmH 15 C+T 334 Q 112 Ochre ND hmH30-2-5 C+T334 Q 11 2 Ochre ND Lost Snu3A hmH30-3-5 C+T676 Q 226 Ochre ND hmH2 C+T 1774 Q 592 Ochre ND hmH 19 G + A 1919 W 640 Amber ND Multiple mutations hmH30-2-4 C+T328 P 110 s ND C+T334 Q 112 Ochre hmH30-3-6 C + T 1934 T 645 I ND C + T 1946 A 649 V C + T 1991 A 664 V Promoter and terminator mutations hmH4 Lac Cin + T -32 ND UV5 hmH7H G +A 2017 hmH30-3-3 G +A 2017 H hmH3-5 G - A 2019 H hmH3O-1-1 C + T 2029 H

* Nucleotide numbering starts from the first baseof the first codon of the mature protein. Amino acid numbering startswith the first amino acid of the mature protein (Thr). ‘ The proteinactivity is indicated by (ND) for none detected, (ST) for detected only in the spot test, and (H) for those which make smaller than normal size halos in the plate complementationassay. RFLPs are restriction fragment length polymorphisms produced by the loss or creation of restriction enzyme recognition sites by the mutations. assay of its product’s function, the ability to rapidly activity to isolate mutations which make smaller than screen for the loss or restoration of a particular func- normal size halos when on plasmids and smaller than tion is certainly advantageous. It was possible to de- normal size plaques when on phage. velop such a screen for the tailspike protein because A small number of mutants were isolated in order its attachment toP22 capsids to make infectious phage to test the feasibility of mutant isolation using the occurs under a wide variety of conditions. The ability plate assay and of deletion mapping of tailspike pro- of the P22 tailspike protein to assemble onto capsids tein mutants. One of the goals of isolating mutants in liquid culture during the growth of tailspike de- made in the plasmid clone of the tailspike protein pendentphage mutants allows the propagation of gene was to find mutants which would be difficult or these defective mutants. In the plate assay which was impossible to isolate on phage such as those with a developed for these studies the level of production or marginal decrease in activity or those which prevent functionality of the tailspike protein expressed from a the propagation of mutant phage by the addition of plasmid carrying gene 9 is measured by the ability of exogenous tailspike protein. Examples of both of these the protein produced to complement P22 capsids in classes of mutants were found. Four of the mutants soft agar to form a halo of lysis around the colonies produce small plaques when they are on phage and making tailspike protein. The size of the halo was a one mutant has a dominant negative phenotype. reliable enough indicator of overall tailspike protein A genetic system was developed for this gene in 646 J. J.Schwarz and P. B. Berget

TABLE 6 AA Suppression patterns of gene 9 nonsense mutants U A G-C Mutant Suppressed 30" 39' -A Amber" hmh3.5 A+ G"-C AG-13.55 kcal/mole hmH5 Q 59 amber-Q + +C +U hmh30.1.1 Q 59 amber-L hmh 30.3.3 A +-c AG-11.7 kcal/mole + AG-13.55 kcal/mole Q 59 amber-S + FA Q 59 amber-Y + A-U hmH19 W 640 amber-+Q - A-U W 640 amber-L - A-U W 640 amber-S - A-U W 640 amber-Y - A-U Ochreb FA hmH30-2-4 P 110 S, Q 112 ochre+ + A-U P 110 S, Q 112 ochre-Y + A-U hmH30-2-5 Q 1 12 0chr-Q + 5". AUGU UUAUG..J' Q 1 12 ochre-Y + FIGURE 4.-Mutations in the presumptive transcription termi- hmH30-3-5 Q 226 ochre-Q + nator. Mutations were isolated on plasmids based on their small Q 226 ochre-Y + halo size in the plate complementation assay. They were moved onto phage P22 by in vivo recombination wherethey produced very hmH2 Q 592 ochre+Q + small plaques. All of these mutations disrupt a potential G-C base Q 592 ochre-Y + pair in the stem of the presumptive transcription terminator. l'tw The ambermutants were transferred to phage P22 by recom- free energies shown are the calculated stabilities of the proposed bination and titered onvarious amber Salmonella suppressor strains mutant stem and loop structure by the method of SALSER(1977) which insert Gln, Leu, Ser or Tyr at both 30" which is the permis- and should be compared to the calculated free energy of the wild sive temperature fortemperature-sensitive tailspike protein mutants type sequenceof -2 1.4 kcal/mole. and 39" which is the nonpermissive temperature. For the amber mutants a (+) indicates that the titer is decreased less than one order of magnitude from that on a suppressor strain which inserts phage h and a plasmid thethreshold for efficient the wild type codon. A (-1 indicates that the titer on these strains recombination was determined tobe at approximately is no higher than the backgroundreversion frequency. 74 bp even though recombination can be detected * Plasmids carrying the ochre mutations were transformed into ochre suppressing E. coli strains which insert Gln or Tyr and the with as little as 20 bp (WATT et al. 1985). The spot tailspike protein activity tested by the plate complementationassay. test for this cross was repeated using larger amounts The ochremutants all hadabout the same small haloon both of phage. Spotting 10' phage onto a lawn was suffi- suppressor strains at 30" and no halo at 39", even when the wild- type amino acid was inserted. cient to detect marker rescuein this cross. So the spot test can correctly map a mutation which is at least 12 which mutations on plasmids could be easily mapped nucleotides from a deletion endpoint.For the majority using deletions carried on phage. Twelve deletions of mutants the spot test is a facile and accurate means were made on plasmids and moved onto phage by of mapping mutations. However for a subset of mu- recombination. This system worked extremely well tants a slightly more elaborate but still simple proce- for all butone mutant which requiredthe useof dure was necessary. heteroduplex deletion mapping. Deletion mapping The spot test was unable to map 8 of the 27 mutants with phage deletions was by two methods: a spot test tested. Seven of theseformed large zones of lysis where the cross is done ona plateand sensitive marker indicative of positive marker rescue with all of the rescue where the cross is done in liquid media. Most deletions. This apparent markerrescue in crosses with mutations required only a rapid spot test. The ability all deletions could have three causes: the mutation is of the spot test to discriminate between mutants lo- not in the region corresponding to any of the dele- cated within a deletion interval and those which are tions, phage carrying the mutation make plaques SO nearby is quite good. The result of the cross between recombination which replaces a deletion with the mu- Dl 1 and hmH30-2-2 show that spotting lo6 deletion tation makes phage which are tailspike independent, phage onto a lawn containing a mutation which is 17 or complementation of phage capsids either intra- or bases removed from the deletion endpointis sufficient extracellularly to make infectious phage. Sensitive to detect marker rescue. Under thesesame conditions marker rescue experiments in which the progeny of however, the cross between Dl and hmH4-1 which these crosses were tested for plaque forming ability are separated by 12 bases failed to produce marker were done to distinguish a plaque forming phenotype rescue. The problem of producing wild type phage by from protein complementation. It was found that the recombination is compounded in this case by there mutations located in the putative transcription termi- being only 64 bases of homology 5' to gene 9 between nator which have enough activity to complement cap- the plasmid and thephage. In recombination between sidsin the plate assay also have enough activity to Tailspike Protein P22 Tailspike 647 make small plaques when on phage. Therefore, for to be as sensitive anindicator of tailspike protein these mutants the apparentpositive marker rescue in activity as the plaque-forming ability of phage because the spot test is the result of both complementation plasmid gene 9 mutations that show up as small halo andtheir phagephenotype. The mutantshmH3-3 mutants in the plate assay formextremely small and HmH30-2-3 were neither able to make plaques plaques when crossed onto phage. The spot deletion when on thephage nor were they able to complement mapping technique seems to be a more sensitive indi- capsids extracellularly in the plate assay,yet they cator because it detected activity in two mutants formed large areas of lysis when crossed in the spot (hmH3-3 andhmH30-2-3) which was not detected in test with phage carrying the deletions in which they the plate assay or by phage phenotype. Therefore the are located. It is possible thesemutants possess an mutants can beseparated into three categories of extremely small amount of activity which, although increasing tailspike protein activity based on the re- insufficient for extracellular complementation in the sults of the plate assay and the spot deletion mapping plate assay, is nevertheless sufficient to complement technique: 1) Those which have no detectable activity the capsid under the conditions of the spot test. The (ND). 2) Those whose activity is only detected in the critical difference between the conditions of the spot spot test (ST). 3) Those whose activity is detected by test and the plate assay is the phage used in the spot the plate assay (H). These categories are indicated in test have had tailspike protein added in vitro to allow Table 5. The two mutants hmH3-3 and hmH30-2-3 them to infect cells once whereas those used in the are the only ones whose mutations are in the protein plate assay are devoid of tailspike protein and require coding region of the gene and have any detectable the cells to supply tailspike protein for infection. So activity. They arealso located very close to each other, during thespot test cells producing the mutant protein codons 563 and 575respectively. It is remarkable that from a plasmid are infected and lysed. Complemen- the four mutants which have enough activity to make tation under these circumstances could be the result small halos and small plaques are all in the presump- of both intracellular complementationand of the large tive transcription terminator and not in protein cod- amounts of mutant protein released bylysis of in- ing sequence. It is possible that mutations in the fected, mutant protein producing cells. The hmH4 protein coding sequencewhich cause marginal activity mutation is located in the lacUV5 promoter which is decreases commensurate withsmall halos are ex- not included in the gene 9 deletions carried on phage, tremely rare. so this mutation produces true positive marker rescue Mutations in the putative transcription terminator with all deletions in sensitive marker rescue. It was were entirely unexpected. It has been suggested in therefore mapped by inference and later confirmed several reports that a RNAstem and loop structure is by DNA sequencing. a barrier to mRNA digestion by exonuclease (WONG The othermutant that was not mapped in the spot and CHANG 1986; BELASCOet al. 1985; GUARNEROS test, hmH10, appeared to produce an extremely low et al. 1982). The mutations all decrease the calculated level of positive marker rescue in the spot test with stability of the stem and loop structure of the mRNA, some deletions and none with others. This same be- so it is possible this structure is a less effective barrier havior was observed with sensitive marker rescue. to mRNA degradation in these mutants because of From the analysis of protein defects of these mutants their lower stability. This could lead to lower expres- (J.J. SCHWARZ andP. B. BERGET,unpublished data) sion of thegene by decreasing the lifetime ofits this behavior appears to be the result of enzymatically message. defective tailspike protein interferingwith attachment A detailed analysis of the structural andfunctional of functional tailspike protein to capsids rather than defects of the missense mutants reported herewill be multiple mutations in the gene. Therefore it was presented elsewhere (J. J. SCHWARZ andP. B. BERGET, mapped with the plasmid heteroduplex deletion map- unpublished data). However, for the purposes of this ping procedure (SHORTLE1983) in which there can discussion it is important to mention that a mutant be nointerference between mutant and functional defective in capsid attachment was isolated by this tailspike protein. The ability of heteroduplex deletion protocol which is a type of mutantthat previous mapping to discriminate whether or not a mutant is screenings had failed to find. In addition,most of the in a deletion interval is impressive. The hmH10 mu- mutants whose isolation is reported in this paper fail tation is separated from the deletion Dl 1 by only 5 to fold into stable trimers at the normally permissive nucleotides but the products of the heteroduplex be- temperature and are thereforedefective in folding or tween Dl l and hmHIO produced functional tailspike stability. However, unlike the temperature sensitive protein in 1 out of the 50 colonies tested. mutants whose mutations are clustered in the middle A crude indication of tailspike protein activity is third of theprotein (VILLAFANEand KING, 1988), provided by the results of the plate assay and the spot these mutations are preferentially located in the car- deletion mapping technique. The plate assay appears boxy-terminal region with more than half of them 648 J. J. Schwarz and P. B. Berget located in the last fifth of the protein. So these muta- GOLDENBERG,D., P. B. BERGETand J. KING, 1982 Maturation of tions probably affect the folding of a domain of the the tail spike endorhamnosidase of Salmonella phage P22. J. Biol. Chem. 257: 7864-7871. tailspike protein other than that defined by the tem- GOLDENBERG, D., H.D. SMITHand J. KING,1983 Genetic analysis perature sensitive mutations and provide the means of the folding pathway for thetail spike protein of phage P22. for a more complete geneticanalysis of this protein’s Proc. Natl. Acad. Sci. USA 80 7060-7064. folding and subunit assembly. GUARNEROS,G., C. MONTANEZ,T. HERNANDEZand D. COURT, 1982 Posttranscriptional control of bacteriophage X int gene Wegratefully acknowledge GEORGE WEINSTOCKand MIRIAM expression from a site distal to the gene. Proc. Natl. Acad.Sci. SUSSKINDfor advice and gift of strains, and MONJULACHIDAM- USA 79: 238-242. BARAM for technical assistance. Research was supported by grants HUMPHREYS,G. O., G. A. WILLSHAW, H. R.SMITH and E. S. from the National Instituteof General Medical Sciences (GM-28952 ANDERSON,1976 Mutagenesis of plasmid DNA with hydrox- and GM-38302) to P.B.B. J.S.was supported by a National Institutes ylamine: isolation of mutants of multi-copyplasmids. Mol. Gen. of Health Institutional Training grant GM-07542 to the University Genet. 145: 101-108. of Texas Health Science Centerin Houston. HURLE, M. R., N. B. TWEEDYand C. R. MATTHEWS, 1986Synergism in foldingof a double mutant of the a- LITERATURE CITED subunit of tryptophan synthase. Biochemistry25: 6356-6360. ISRAEL,J. V., T. F. ANDERSONand M. LEVINE,1967 In vitro ALBER,T., andJ. A. WOZNIAK,1985 A genetic screen for muta- morphogenesis of phage P22 from heads and base-plate parts. tions that increase the thermal stability of phageT4 lysozyme. Proc. Natl. Acad. Sci. USA 57: 284-291. Proc. Natl. Acad. Sci. USA 82: 757-750. IWASHITA,S., and S. KANEGASAKI,1973 Smooth specific phage BELASCO,J. G., J. T. BEATTY,C. W. ADAMS, A.VON GABAINand adsorption:endorhamnosidase activity of tail partsof P22. S. N. COHEN, 1985 Differential expression of photosynthesis Biochem. Biophys. Res. Commun. 55: 403-409. genesin R. capsulata resultsfrom segmental differences in IWASHITA,S., and S. 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COULSON,1977 DNA sequenc- Biochim. Biophys. Acta 407: 365-375. ing with chain-terminatinginhibitors. Proc. Natl. Acad. Sci. GOLDENBERG,D., and J. KING, 1982 Trimeric intermediate in the USA 74: 5463-5467. in vivo folding and subunitassembly of the tail spike endorham- SAUER, R. T., W. KROVATIN, A. R. POTEETEand P. B. BERGET, nosidase of bacteriophage P22. Proc. Natl. Acad.Sci. USA 79: 1982 Phage P22 tail protein: gene and amino acid sequence. 3403-3407. Biochemistry 21: 581 1-5815. P22 Tailspike Protein 649

SCHMITZ,A., C. COULONDREand J. H. MILLER,1978 Genetic using phosphorothioate-modified DNA. Nucleic Acids Res. 13: studies of the lac repressor. V. Repressors which bind operator 8765-8785. more tightly generated by suppression and reversion of non- VILLAFANE,R., and J. KING,1988 Nature and distribution of sites sense mutations. J. Mol. Biol. 123: 431-456. of temperature sensitive folding mutations in the gene for the SHORTLE,D., 1983 A genetic system for analysis of staphylococcal P22 tailspike polypeptide chain. J. Mol. Biol. 204: 607-619 nuclease. Gene 22: 181-189. WATT, V. M., C. J. INGLES, M. S. URDEA and W. J. RUTTER, SIGNER,E. R., and J. WEIL,1968 Recombination in bacteriophage 1985 Homology requirementsfor recombination in Esche- X. 1. Mutants deficient in general recombination. J. Mol. Biol. richia coli. Proc. Natl. Acad. Sci. USA 82: 4768-4772. 34 261-271. WEINSTOCK,G. M., 1977 Genetic and physical studies of bacteri- SMITH,D. H., P. B. BERGETand J. KING, 1980 Temperature- ophage P22 genomes containing translocatable drug resistance sensitive mutants blocked in the folding or subunit assembly of elements. Thesis, MIT, Cambridge, Mass. the bacteriophage P22 tail-spike protein. I. Fine-structure map WEINSTOCK,G. M., M. SUSSKINGand D. BOTSTEIN, ping. Genetics 96: 331-352. 1979 Regional specificity ofillegitimate recombination by the SMITH,H. O., AND M. LEVINE,1967 A phage P22 gene control- translocatable ampicillin-resistanceelement Tn I in the genome ling integration of prophage. Virology 31: 207-216. of phage P22. Genetics 92: 685-710. SUSSKIND,M. M., D. BOTSTEINand A. WRIGHT, 1974 WINSTON, F., D. BOTSTEIN and J. H. MILLER, 1979 Superinfection exclusion by P22 prophage in lysogensof Characterization of amber and ochre suppressors in Salmonella Salmonellatyphimurium. 111. Failure of superinfecting phage typhimurium. J. Bacteriol. 137: 433-439. DNA to enter sieA+ lysogens. Virology62: 350-366. WONG,H. C., and S. CHANG,1986 Identification of a positive SUSSKIND,M. M., A. WRIGHT and D. BOTSTEIN, 1971 retroregulator that stabilizes mRNAs in bacteria. Proc. Natl. Superinfection exclusion by P22 prophage inlysogens of Acad. Sci. USA 83: 3233-3237. Salmonella typhimurium. 11. Genetic evidence for two exclusion YOUDERIAN,P., and M. M. SUSSKIND,1980 Bacteriophage P22 systems. Virology 45: 638-652. proteins specified by the region between genes 9 anderf. SUT-I'CLIFFE,J. G., 1979 Complete nucleotide sequence of the Virology 107: 270-282. Escherichia coli plasmid pBR322. Cold Spring Harbor Symp. ZAGURSKY,R. J., and M.L. BERMAN,1984 Cloning vectors that Quant. Biol. 43: 77-90. yield high levels of single-stranded DNA for rapid DNA se- TAYLOR,J. W., J. OTT and F. ECKSTEIN,1985 The rapid gener- quencing. Gene 27: 183- 19 1. ation of oligonucleotide-directed mutations at high frequency Communicating editor: G. MOSIG