Perspectives. Anecdotal, Historical and Critical Commentaries on Genetics. Thirty Years Ago in GENETICS: Prophage Insertion Into Bacterial Chromosomes

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1 Copyright by the Genetics Society of America Perspectives Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove Thirty Years Ago in GENETICS: Prophage Insertion Into Bacterial Chromosomes Allan M. Campbell Department of Biological Sciences, Stanford University, Stanford, Calqornia VER 30 years ago, a model for insertion of X 0 prophage into the host chromosome during lysogenization was first proposed (CAMPBELL 1961a). The model was developed during the writing of a review on episomes, major inputs being experimental work on prophage gene order by CALEF and LICCIAR- DELLO (1960), studies on the genetic content of Xgul phages (culminating in a March, 1963 paper in GE- NETICS) and some provocative ideas of FRANK STAHL S about chromosome circularity in prokaryotes. Retrospectively, the event can be viewed as one incident in the understanding of chromosome structure, and as one argument for a position that has now been accepted so completely that it may seem surprising there was ever dissent. The issue was whether the chromosome is unidimensional (linear or circular) and, if so, whether added elements such as proviruses are integral parts of it. The two questions are closely intertwined. Had it transpired (as was frequently supposed prior to 1962, e.g., JACOB and WOLLMAN 1961) that a prophage could establish a permanent association with a chromosomal site by some means other than intercalation, it would have been illogical to deny that chromosomal genes might employ the same mode of association. Whereas classical linkage analysis and cytogenetics provided definitive evidence that genes were linearly disposed along the length of the chromosome, and fine structure genetics was equally convincing as to the linearity of very small, gene-sized segments, the physical studies of the time did not exclude models of chromosome structure where DNA genes were attached laterally to a protein backbone or joined end to end by protein connectors (FREESE 1958) or where chromosomes were composed of parallel DNA double helices held in register by lateral protein bonds (CAV- ALIERI and ROSENBERC 1961). The latter model needed only minor extensions to accommodate side- by-side synapsis of a prophage to a segment of homeologous chromosomal DNA (JACOB and WOLLMAN 1961). Autoradiography of bacterial chromosomes, although indicating a circular DNA-containing structure, lacked the necessary resolution to settle the issue; and traditional biochemistry left open the possibility that lateral elements attached noncovalently might be lost during purification. Although various physical studies in the 1970s encouraged the current view that each chromosome (prokaryotic or eukaryotic) contains a single DNA molecule, joined throughout by phosphodiester bonds, the proof awaited restriction mapping, chromosome walking, and sequencing. Current techniques of course also demonstrate that added elements (proviruses, transposons, integrating plasmids) are part of the continuity of the chromosome. However, it was already possible 30 years ago to provide convincing genetic arguments on this score, and bacteriophage X was the first element to which such arguments were applied. The goal of the genetic experiments was to construct an integrated map of a chromosome containing an added element. The prerequisites were an element marked with several mu- tations and a chromosome that was genetically marked in the flanking DNA. It was shown early on that X prophage could be mapped close to the gal and bio operons of Escherichia coli K-12, in which mutations were available. In X, plaque morphology mutations could be used; or more conveniently, conditionally lethal mutations. Hfr X F- bacterial crosses consistent with intercalation of X between gal and the fairly distant trp locus (CALEF and LICCIARDELLO 1960) were followed by P1 transductions with the closely flanking loci gal and bio (ROTH- MAN 1965). Selection for mutations inactivating nearby chromosomal genes provided nested sets of deletions that penetrate into and through the pro- Genetics 133: (March, 1993)

2 434 A. M. Campbell phage, first in a X-related phage (FRANKLIN, DOVE and mutants plated only on some K-12 strains, the same YANOFSKY 1965), then in X itself (ADHYA, CLEARY ones on which my X mutants plated. BOB thought this and CAMPBELL 1968). might be a general method for finding mutations Mostof the above experiments came some time whose products were essential for phage development. after the initial proposal for X insertion. The earlier This encouraged me to isolate many more mutations, evidence (besides the workof CALEF and LICCIAR- arrange them into complementation groups, test the DELLO) came from examination of Agar specialized effect of E. coli suppressors on mutant phage growth, transducing phages, which had been discovered a few and isolate thermosensitive mutants for comparison years earlier by MORSE, LEDERBERG and LEDERBERG (CAMPBELL 1961b). When I sent my manuscript to (1956), and from segregation patterns of double ly- BOB, he wrote back, Great minds run in the same sogens containing one normal X and one Xgal in tandem (CAMPBELL 1963a). The interpretation of the tracks, and explained that he had mutants of T4 for several months. been isolating ts Xgal results was logically equivalent to the topological mapping used with deletions (BENZER 1959), the basic assumption being that each Xgal isolate repre- Later, in a semihistorical note (EDGAR 1966), BOB raised the question of why there was such a long time lag between the isolation of ts mutations in Neurospora sented a connected segment of the lysogenic chromosome. The enzymology behind the rare excisions that produce Xgul s is still unknown. It was clear 30 years ago that Xgal s have a large number of different endpoints, both at the phage and the bacterial end, and that specific Xgal types are probably generated at the time of excision, rather than earlier in the development of the bacterial clone (CAMPBELL 1963b). The detailed analysis of Xgal phages required the availability of a large collection of mutations distrib- uted over the phage genome; and to be practical, it required that prophage genotypes be easy to score. This approach was greatly facilitated by the use of conditional-lethal mutants, which allowed scoring by spot tests rather than by making lysates and checking plaque morphology. A digression on the discovery of conditional lethals in X: they were first observed in 1958, quite by accident. For the studies on Xgul gene content, I wanted mutations at many sites of the X genome, and decided it might help to induce small-plaque mutations by SOS mutagenesis (WEIGLE 1953). The standard strain for plating X was C600, a K-12 derivative. I isolated two small-plaque mutants and made lysates of them. When I assayed these lysates, I had run out of C600 and used instead a second X-sensitive K-12 strain, W3350. No plaques were visible. Checking, I found that the mutants formed plaques only on C600, not on W3350. I had no idea why the host specificity, but it made the mutants very useful for the kind of mapping studies I contemplated; and it proved simple to collect more of them. From the first 15 mutations, it was clear that they mapped to many parts of the X genome, and that most mutant pairs complemented in mixed infection. I sent some of the mutants to JEAN WEIGLE at CalTech, where my own work on X transduction had started. In 1959, BOB EDGAR told me about some results of DICK EPSTEIN S T4. on At CalTech, EPSTEIN had looked for T4 mutations that allowed plaque formation on K- 1 2 but not on the standard T4 plating host, E. coli B, and had later found that such amber crassa (HOROWITZ and LEUPOLD 1951) and rediscovery of the principle with T4. In my case, rediscovery was not necessary. The Neurospora experiments came to mind in my initial conversation with BOB, and I suspect that he may have had a subliminal recollection as well. At any rate, as noted elsewhere (BERG 1990), my own explanation for the time lag is that neither BOB nor I would have proceeded in these studies without the example of BENZER S work on fine structure genetics, which raised our consciousness of the manner inwhich conditional lethals could be most constructively classified and analyzed. The T4 group, led by EPSTEIN and EDGAR, proceeded to a comprehensive analysisof mutant function, whereas I employed the mutations primarily as genetic markers. In the late 1960s, many members of the X group carried the physiological analysis of my mutants to a similar level of sophistication, apparently stimulated lessby my original paper or the T4 work than by the thesis workof one of my students (BROOKS 1965), which initiated analysis of X replication genes. BROOKS paper, coming at a time when X regulation seemed ripe for investigation, found an audience prepared to respond. Returning to X insertion, the model postulated that the ends of the linear phage genome became joined prior to insertion, thereby allowing a single crossover to insert the prophage with the observed cyclic permutation of gene order. Direct evidence for endjoining, by ligation of sticky ends, soon became available (KAISER and Wu 1968). A few years later, specialized transducing phages provided direct molecular evidence of DNA insertion. When the DNA from a Xgal of known gene content formed a heteroduplex with X DNA, the physical extent of the gal substitution could be used to locate genes on a DNA map. More fundamentally, the results argued strongly that phage DNA and bacterial DNA are covalently joined, and that no non-dna linkers separate prophage from host DNA (DAVIDSON and SZYBALSKI 197 Eventually, 1). Xgal and Xbio specialized transducing phages provided junction fragments

3 Perspectives 435 go/ XY ABCD cl mi h ABCD fv t - C OF LYSOGENIC BKlEIWJM FIGURE 1.-Prophage insertion as proposed in CAMPBELL (1961a). The original legend reads "Possible mechanisms of lysogenization by reciprocal crossing over between a circular phage chromosome and a linear bacterial chromosome. Arrows indicate rare points of breaking and joining in the formation of transducing lambda. The genes ABCD are hypothetical and indicate a small region of homology between host and phage. X and Y are unspecified bacterial genes." Thirty years later, ABCD is identified as the 15-mer "GCTTTTTTATACTAA" and X and Y could be chld and pgl. whose sequences define the insertion event with precision (LANDY and ROSS 1977). Genetic control: As originally formulated, the insertion model postulated crossing over between a unique sequence of the circular phage genome and a homolog on the bacterial chromosome (Figure 1). Thus it used the known process of homologous recombination to explain the unknown mechanism of prophage insertion. The reasonable assumption that such a crossover should require the RecA protein was soon tested and proven incorrect. The work led instead to the discovery that RecA is needed for repressor destruction following SOS induction (BROOKS and CLARK 1967). Another prediction was that increasing the extent of homology should increase the rate of insertion; instead, even extensive homology had little effect if repressor was present, indicating that phage- a coded protein might be required. A search for mutations affecting insertion identified single a phage gene (int); later a second gene (xis) was identified whose product was needed (in addition to Int) for excision from the chromosome. Two host-coded proteins (the heterodimeric integration host factor IHF and the Fis protein) were later shown to play a role in the integrase reaction, both by in vivo genetics and in vitro biochemistry (reviewed in CRAIG 1988). Enzymology: This genetic work set the stage for the identification of the int gene product, which proved to be a site-specific recombinase (NASH 1981). A number of elegant biochemical experiments defined the reaction mechanism. Integrase causes a twostrand exchange at a precise nucleotide position by way of a transient DNA-protein bond on a tyrosine residue of the integrase, to generate a Holliday crossbridge. Branch migration across a 7-bp segment that is identical in both partners occurs next, then the Holliday structure is resolved by exchange between the other two strands. The result is a precise crossover. The whole reaction proceeds in a concerted manner, requiring neither an external energy source nor repair synthesis. The substrate requirements for the reaction are a 21-bp segment from the bacterial site (attb) and a 235-bp segment from the phage (attp). The in vitro reaction works best when attp is supercoiled. The initial step in integration is binding of Int to attp, followed by association of the complex with at&. The reaction is then catalyzed by the C-terminal domains of integrase molecules associated with DNA sequences (core sites) symmetrically around the 7-bp overlap segment. Binding of integrase to core sites is enhanced by high affinity binding of the N-terminal integrase domain to arm sites in the flanking DNA of attp bp from the crossover point, as well as by proteinprotein interactions. Proper positioning of integrase molecules is facilitated by binding of IHF, a DNA-bending protein, at specific sequences between the core and arm sites. Xis and Fis are also DNA-bending proteins, which promote formation of the complex in the excision reaction. The system provides an instructive model for DNA looping (KIM and LANDY 1992). Soon after the discovery of excisionase, a physical chemical question was raised (DOVE 1970): if the insertion reaction comes about simply through catalysis of DNA recombination by an enzyme (integrase), then the second law of thermodynamics requires that the catalyst work equally well in both directions, approaching an equilibrium position depending solely on the energy contents of reactants and products. If Xis were a second catalytic component, it could not change the equilibrium position. The thermodynamic driving force has still not been identified with certainty. However, the question is circumscribed by the demonstration that branch migration proceeds in the same direction in excision as in insertion (NUNES- DUBY, MATSUMOTO and LANDY 1987). Therefore, excision is not the true reversal of insertion; but the failure of integrase to promote the direct reversal efficiently remains unexplained. Regulation: Both insertion and excision are efficient. Insertion takes place in almost every cell surviving infection; and even transient derepression can induce excision in most cells of a lysogenic culture (WEISBERG and GALLANT 1967). Nevertheless, only some years after the discovery of integrase and excisionase was it discovered that their expression is differentially regulated. It was known early on that the two genes could be transcribed from the major leftward promoter (pl), which was turned on following either infection of a sensitive host or derepression of a lysogen. Obligatory coexpression of these two genes seemed ill suited to the dissimilar enzyme requirements for insertion us. excision. An indication that integrase might have a

4 436 A. M. Campbell second promoter came from characterization of rare lysogens with X inserted into the trpc gene rather than at its normal location (SHIMADA and CAMPBELL 1974). SHIMADA found that such lysogens transcribe the adjacent trpb gene at a low constitutive rate, and that this transcription is eliminated by internal prophage deletions penetrating int. Selection for a high rate of trpb expression produces phage mutants (int-c) that transcribe int even as prophages. Later sequencing showed that the int-c mutation improves the -1 0 region of the normal int promoter (PI). The importance of PI became apparent only after the discovery that it is strongly stimulated by the the X CII protein (reviewed in ECHOLS and GUARNEROS 1983). (SHIMADA S int-c mutant is not stimulated by CII, for reasons that remain obscure.) It was known that CII is the principal macromolecular effector driving an infected cell toward lysogeny rather than lysis, by turning on transcription of the repressor gene cz. Stimulation of PI by the same effector ensures a high rate of integrase production in cells surviving infection but not in those that lyse. Deletion analysis located the CII-responsive element very close to PI (HEFFER- NAN, BENEDIK and CAMPBELL 1979); we now know it is the -35 region of PI. On the other hand, when a lysogen is derepressed, transcription from pl results in coordinate expression of int and xis, directing the reaction toward excision. The system turned out to have an additional refinement. Coordinate expression of int and xis is desirable following derepression of a lysogen but not in an infected cell before commitment to lysis or lysogeny. It turns out that very little integrase is formed from pl transcription of infecting DNA, because pl mrna degradation is initiated by host RNAse IIImediated cleavage at a site (sib) downstream from int. In the inserted prophage, sib is separated from int because it lies on the opposite side of attp; thus the pl transcript from a derepressed lysogen is more stable (ECHOLS and GUARNEROS 1983). X insertion thus became the first well characterized example of a programmed DNA rearrangement. Impact on other research areas: As a demonstrated specific DNA rearrangement, X insertion provided encouragement for the belief that such rearrangements might be used elsewhere. For example, DREYER and BENNETT (1965) invoked X insertion as a precedent for their novel proposal (later proven correct in principle) that gene rearrangement underlies the determination of immunoglobulin specificity. X insertion also influenced work on the insertion of transposable elements, including phage Mu and retroviruses. As the mechanism turned out to be different, some of the parallels proved misleading. Transposons insert from linear rather than circular substrates, and no homology is needed at the target site. Retroviroiogists retained an affection for a circular integration substrate like X s until quite recently, and I have enjoyed an intermittent dialog with them over the years. In an early edition of The Molecular Biology of the Gene, JIM WATSON (1965) bravely diagrammed the insertion of another cancer virus (polyoma) as a X-like event. In retroviral infection, a minor fraction of the viral DNA prior to insertion is in circular form, resulting either from ligation of the terminal repeats or from recombination between them. Some retroviruses can use the genetic sequence of the ligated ends as an integration substrate (PANGANIBAN and TEMIN 1984); however, the major pathway, strongly supported by in vitro data, is through insertion of the linear molecule into the target site (BROWN et al. 1987). Evolution and natural variation: X has many natural relatives (collectively known as lambdoid phages), which have a common genetic map and presumed common ancestry but exhibit extensive variation in specific traits. Some lambdoid phages (such as X and 434) insert at a common chromosomal site and encode interchangeable integrase and excisionase proteins. Others insert at various sites and encode proteins with corresponding specificities. As all their integrase genes are related, this raises the question of how specificities have changed and why natural selection might have favored variants with altered specificities. The simplest answer to the why question is frequency-dependent selection (which has often been invoked for other host-parasite interactions). When two phages have the same specificity, each can sometimes expel the other when it inserts. A rare variant with a new specificity is never expelled by other phages-an advantage that lasts only until it becomes as common as other types. The how question has been approached by studying the properties of natural phages with overlapping specificities. The best examples are X us. HK022, which share the same specificity for excisionase and arm binding but differ in core binding; and phage 21 vs. defective element e14, which insert at the same site but differ in recognition specificity, so that neither one can expel the other from the chromosome. Current research is defining the molecular determinants of specific recognition in the two cases (CAMPBELL 1992; NACARAJA and WEIS- BERG 1990). While X inserts into intergenic DNA, some lambdoid phages insert within structural genes for proteins or trnas. In those cases, the phage DNA includes on one side of the crossover point a duplicate (either precise or imprecise) of the 3 end of the target gene. Thus, the lysogen expresses a functional gene whose 3 end is phage-derived. The simplest historical scenario is that the ancestral phage arose in a strain with a preexisting duplication of the gene in question; and that the phage later picked up a short segment of flanking host DNA by the same mechanism used in

5 Perspectives 437 Agul formation, thus enabling it to lysogenize other strains where the gene was unique (CAMPBELL 1992). In a wider context, X integrase can be placed by DNA and protein homology into a large family of sitespecific recombinases, the integrase family, which is represented in both prokaryotes and eukaryotes. (A second family, responsible for DNA inversions and transposon resolution, is not detectably related to the integrase family.) All of the prokaryotic elements that insert by site-specific recombination use enzymes that belong to the integrase family: and the existing data indicate that, despite extensive sequence divergence, they all have similar attributes, such as a 7-bp overlap segment. A currently popular notion is that all these elements are derived from an ancestral phage that inserted into the anticodon loop of a trna gene (where a unique 7-bp sequence is flanked by short inverted repeats). This fits with the widespread use of trna insertion sites by elements with diverse hosts. Work currently underway in many laboratories should fill some of the gaps in the evolutionary picture. I am grateful for continuous support since 1959 from the National Institute of Allergy and Infectious Diseases, grant AI LITERATURE CITED ADHYA, S., P. CLEARY and A. CAMPBELL, 1968 A deletion analysis of prophage lambda and adjacent genetic regions. Proc. Natl. Acad. Sci. USA 61: BENZER, S., 1959 On the topology of the genetic fine structure. Proc. Natl. Acad. Sci. USA 45: BERG, D. E., 1990 Conditional mutations in procaryotes, pp in The Bacterial Chromosome, edited byk. DRLICA and M. RILEY. American Society for Microbiology, Washington, D.C. BROOKS, K., 1965 Studies in the physiological genetics of some suppressor-sensitive mutants of bacteriophage X. Virology BROOKS, K., and A. CLARK, Behavior of X bacteriophage in a recombination deficient strain of Escherichia coli. J. Virol. 1: BROWN, P. O., B. BOWERMAN, H. E. VARMUS and J. M. BISHOP, 1987 Correct integration of retroviral DNA in vitro. Cell 49: CALEF, E., and G. LICCIARDELLO, 1960 Recombination experiments on prophage host relationships. Virology 12: CAMPBELL, A. M., 1961a Episomes. Adv. Genet. 11: CAMPBELL, A., 1961b Sensitive mutants of bacteriophage X. Virology 14: CAMPBELL, A,, 1963a Segregants from lysogenic heterogenotes carrying recombinant lambda prophages. Virology 20: CAMPBELL, A,, 1963b Distribution of genetic types of transducing lambda phages. Genetics 48: CAMPBELL, A,, 1992 Chromosomal insertion sites for phages and plasmids. J. Bacteriol. 174: CAVALIERI, L. F., and B. H. ROSENBERG, 1961 The replication of DNA Changes in the number of strands in E.coZi DNA during its replication cycle. Biophys. J. 1: CRAIG, N. L., 1988 The mechanism of conservative site-specific recombination. Annu. Rev. Genet. 22: DAVIDSON, N., and W. SZYBALSKI, 1971 Physical and chemical characteristics of lambda DNA, pp in The Bacteriophage X, edited by A. D. HERSHEY. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. DOVE, W., 1970 An energy level hypothesis for X prophage insertion and excision. J. Mol. Biol. 47: DREYER, W. J., and J. C. BENNETT, 1965 The molecular basis of antibody formation: a paradox. Proc. Natl. Acad. Sci. USA ECHOLS, H., and G. GUARNEROS, 1983 Control of integration and excision, pp in Lambda 11, edited by R. W. HENDRIX, J. W. ROBERTS, F. W. STAHL and R. A. WEISBERG. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. EDGAR, R. S., 1966 Conditional lethals, pp , in Phage and the Origins ojmolecular Biology, edited by J. CAIRNS, G. S. STENT and J. D.WATSON. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. FRANKLIN, N. C., W. F. DOVE and C. YANOFSKY, 1965 The linear insertion of a prophage into the chromosome of E. coli shown by deletion mapping. Biochem. Biophys.Res. Commun. 18: FREESE, E., 1958 The arrangement of DNA in the chromosome. Cold Spring Harbor Symp. Quant. Biol. 23: HEFFERNAN, L., M. BENEDIK and A. CAMPBELL, 1979 Regulatory structure of the insertion region of bacteriophage X. Cold Spring Harbor Symp. Quant. Biol. 43: HOROWITZ, N. H., and 0. LEUPOLD, 1951 Some recent studies bearing on the one gene-one enzyme hypothesis. Cold Spring Harbor Symp. Quant. Biol. 33: JACOB, F., and E. WOLLMAN, 1961 Sexuality and the Genetics of Bacteria. Academic Press, New York. KAISER, A. D., and R. Wu, I968 Structure and function of DNA cohesive ends. Cold Spring Harbor Symp. Quant. Biol. 33: KIM, S., and A. LANDY, 1992 Lambda Int protein bridges between higher order complexes at two distinct chromosomal loci attl and attr. Science LANDY, A,, and W. Ross, 1977 Viral integration and excision. Structures of the lambda att sites. Science 197: MORSE, M., E. LEDERBERG and J. LEDERBERG, 1956 Transduction in Escherichia coli K-12. Genetics41: NAGARAJA, R., and R. A. WEISBERG, 1990 Specificity determinants in the attachment sites of bacteriophages HK022 and A. J. Bacteriol. 172: NASH, H. A., 1981 Integration and excision of bacteriophage lambda: the mechanism of conservative site-specific recombination. Annu. Rev. Genet. 15: NUNES-DUBY, S. E., L. MATSUMOTO and A. LANDY, 1987 Sitespecific recombination intermediates trapped with suicide substrates. Cell 50: PANGANIBAN, A. T., and H. M. TEMIN, 1984 Circles with two tandem LTRs are precursors to integrated retrovirus DNA. Cell 36: ROTHMAN, J. L., 1965 Transduction studies on the relation between prophage and host chromosome. J. Mol. Biol. 12: SHIMADA, K., and A. CAMPBELL, Int-constitutive mutants of bacteriophage lambda. Proc. Natl. Acad. Sci. USA71: WATSON, J. D., 1965 The Molecular Biology of the Gene. W. A. Benjamin, New York. WEIGLE, J., 1953 Induction of mutations in a bacterial virus. Proc. Natl. Acad. Sci. USA 39: WEISBERG, R., and J. GALLANT, 1967 Dual function of the X prophage repressor. J. Mol. Biol