Genetica 86: 259-267, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Lambdoid phages as elements of bacterial genomes (integrase/phage21! Escherichia coli K-12ficd gene)

A. Campbell, S. J. Schneider & B. Song Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA

Received and accepted 10 March 1992

Abstract

The lambdoid phages are a group of related temperate bacteriophages that lysogenize by site-specific recombination with the bacterial chromosome. Various members of the group have different specific chromosomal insertion sites, despite the fact that the enzymes catalyzing the insertion (integrases) appear to be all descended from a common ancestor. Insertion sites are not located randomly on the E. coli chromosome but are restricted to one segment of the map; also, most prophages are oriented in the same direction along the chromosome. Lambdoid phage 21 inserts within the isocitrate dehydrogenase gene and introduces an alternative 165 bp 3' end for that gene. A defective element (el4) inserts at the same position. We suggest that this mode of insertion arose from insertion of an ancestral phage to the fight of icd which then picked up part of the icd gene by abnormal excision and speculate that, at an earlier time, phages may have arrived at their present locations by a process of chromosomal walking.

Accessory DNA elements have evolved at least two distinct mechanisms for inserting themselves into chromosomes. Transposable elements generate, during insertion, oligonucleotide duplications of their target sites. Typically they show relatively little specificity for the target sequence but specific recognition of transposon termini. Insertion by con- servative site-specific recombination, on the other hand, proceeds by breaking and joining of identical oligonucleotide sequences pre-existing in a circular extrachromosomal element and the chromosome and is typically specific for both element and chro- mosomal site. These two classes of inserting ele- ments raise many common questions as to the se- lective pressures acting on element and host.

Among the temperate phages that lysogenize by DNA insertion, some (such as phage Mu-1) use the same enzymology employed by non-phage trans- posable elements. Others, like the lambdoid pha- ges, insert with high preference at unique sites (specific for the particular phage) by use of site- specific recombinases. Although lambdoid phages generally do not move around within genomes once inserted, they can insert at low frequency into dif- ferent sites; over evolutionary time, the lambdoid

family has produced types capable of preferential insertion at various locations.

Our major focus will be on the evolution of new insertion specificities by the phage and some possi- ble interactions with host evolution. We start with a summary of the major features of lambdoid phage evolution and population biology, as inferred from the comparative study of natural isolates.

The lambdoid phages

The lambdoid phages all infect Escherichia coli or closely related bacteria. They share a common ge- netic map and recombine to produce viable hybrids. All have life cycles similar to h's. Linear phage DNA injected on infection is quickly circularized and may either replicate (lytic infection) with even- tual formation of new viral particles followed by lysis of the cell or may insert into the chromosome (lysogenic cycle) with concurrent repression of lytic functions. Among lambdoid phages, there is great variation in the specificities of repression, insertion, host cell attachment and gene regula- tion. Genes encoding DNA recognition proteins are

260

generally located close to their target sites; e.g., the replication origin lies within the structural gene O for the specific initiation protein.

No natural lambdoid phage has been entirely sequenced, but the sequence is known for a 'labora- tory lambda' derived from natural lambda (Daniels et al., 1983). Extensive blocks of sequence have been determined for some other lambdoid phages. Overall patterns of sequence relatedness are also deducible from heteroduplex analyses (Highton et al., 1990).

Comparison of any two lambdoid phage genomes reveals extreme base sequence divergence in some segments and near-identity in others. Ex- treme divergence generally accompanies altered specificities for such properties as repression, inser- tion, gene regulation, host cell attachment, or repli- cation. If we consider, for example, the four lambdoid phages h, 21, P22 and 434, k and 434 prophages insert at the same bacterial site and make similar, functionally interchangeable integrase pro- teins. Sequence comparison shows that 21 integrase is more closely related to k and 434 integrases than is P22 integrase. An evolutionary tree based on integrases does not apply to the whole phage genome, however. In repressor specificity, k and 434 are unique, whereas 21 and P22 closely resem- ble each other. In specificity for attachment to the cell surface (mainly a function of the terminal pro- tein J of the tail fiber) k and 21 are very similar, 434 has a different specificity, and P22 (whose host of origin is Salmonella typhimurium) not only has its own specificity but also a very different tail mor- phology, and packages DNA by a headful mecha- nism rather than by cutting at specific sites.

The distribution of homologies and isospecifici- ties among lambdoid phages is compelling evi- dence that recombination between these phages is a frequent natural event - so much so that it is tenable to regard the entire array of lamddoid phages as a biological species drawing functional genetic mod- ules from a common gene pool (Campbell & Bot- stein, 1983; Campbell, 1988). Even within nearly homologous segments, the sequence patterns sug- gest past exchanges; and within largely heterolo- gous segments, very short segments of similar se- quence may have experienced multiple recombina- tion events (Baker et al., 1991). The gene pool includes phages, prophages and the defective pro- phages that litter many bacterial chromosomes.

Probably the most common natural encounters where recombinants may arise are between infect- ing phages and resident prophages, active or defec- tive.

Such a species concept implies not only that recombination among natural lambdoid phages is common, but also that gene flow from other sources into the phage population is rare. Here direct dem- onstration is difficult. Some distant homologies be- tween phage and host gene are known, and it is generally assumed that phage genes were derived originally from host genes serving similar func- tions. A good case can also be made that genes have occasionally been transferred between phages that are largely unrelated (Haggard et al., 1992).

The attachment site

One segment of the phage genome whose origin poses special questions is the insertion site on the phage. In lambda, the recognition signals for inser- tion span 240 bp centered on a 15 bp segment that is identical between phage and host and includes the crossover point (Weisberg & Landy, 1983). The inserted prophage thus is bracketed by a 15 bp repeat in direct orientation. In other lambdoid phages, the repeat is even longer, although only 7 bp of precise homology are required by the reaction mechanism (Leong et al., 1985).

Such flanking direct repeats are nearly universal attributes of inserted DNA elements, although their mechanism of origin varies. Transposons (includ- ing retroviruses and Mu-1) generate oligonucleo- tide repeats of target DNA during insertion. Some plasmids, such as F, use homologous recombina- tion within insertion sequences common to plasmid and host (Davidson et al., 1975). With lambdoid phages, the repeat is not generated during insertion but pre-exists. This raises the question of how phage and bacterial sequences came to resemble each other. Three possibilities can be entertained. (a) chance: In a random DNA sequence where the four bases are equally common, an arbitrary 15-mer should occur about once every 10 9 bases. The chance that the 4000 kb E. coli chromosome would contain a specific 15-mer present in lambda is therefore about one in 250. For phages with longer repeats, the probability is infinitesimal: no 25-mer in the whole 50 kb of lambda DNA is likely to

261

A . i ccl "

X

@ icd l I pre 21

.............. P

i cd

G |

icd . 1 pre 21 'icd I I

C. icd

icd ,- ' icd 21 ' icd

Fig. 1. Possible origin of 21 att. (A) Insertion of an ancestral phage ('pre 21 ') to the fight of icd. Abnormal excision generated a pre 1 icd phage; - (B) Insertion of this pre 21 ied phage by homologous recombination; - (C) Insertion of present-day 21, where insertion is not through homology but through site-specific recombination at the phage-iedjunction. Present-day 21 may also have deleted some bacterial DNA between the 3' end of ied and the insertion site of pre-21. The drawing is not to scale. Phage 21 is about 43 kb; icd is 1.3 kb.

262

occur in the chromosome by chance alone. (b) learning to look alike: Phages can insert at low frequency into sites that lack an exact match, even in the critical 7 bp segment where branch migration takes place. Perhaps the original insertion at a given bacterial site came about by this means, after which the phage by mutation, imprecise excision or gene conversion came to resemble the site more closely. The phage can acquire the 7 bp segment in one cycle of insertion and excision (Weisberg & Landy, 1983). Imprecise, int-catalyzed recombination can also generate other changes that alter DNA-DNA recognition at the crossover point (Leong et al., 1984). (c) common origin: The phage site might be derived from the host or vice versa.

We currently study lambdoid phage 21, where a common origin from the host is strongly indicated.

Insertion within a structural gene

Phage 21 inserts within the structural gene for iso- citrate dehydrogenase (ica9 in a 10 bp segment that is identical between phage and host and lies 165 bp from the 3' end of icd (S. Schneider and A. Campbell, unpublished data). The icd function is not destroyed by the insertion, because the phage encodes an alternative 3' end of icd, which differs from the host sequence in about 7% of its nucleo- tides. In some E. coli strains (such as K-12), this site in icd is occupied by a defective element (el4), which includes its own version of the 3' end of icd (which differs from both 21 and host by about 7 %). Strains that have lost e 14 have normal icd function, and phage 21 still can insert. Clearly the insertion sites of both 21 and e l4 are derived from host DNA.

The conclusion is not unique to phage 21. Sev- eral other phages (P4, phi R73, P22, hDLP12, Hplcl of Hemophilus) or inserting plasmids (SSVI of Sulfolobus, SLP 1 of Streptomyces, pMEA100 of Nocardia, pSE101 and pSE211 of Saccharopoly- spora) insert into known or putative tRNA genes and likewise include a 3' end sequence within the phage DNA. (Reiter et al., 1989; Lindsey et aL, 1989; Brown et al., 1990; Sun et al., 1991). These cases differ from phage 21 in that the sequences are generally shorter and identical rather than similar. Because the sequences are identical, the primary

structures of the tRNAs specified by lysogens are identical to those in non-lysogens, unlike the case with icd. The closest parallel to phage 21 is with the Atlas phages (Stolzfus, 1991) which insert near the trp operon at 28 min. Atlas prophages (frequently defective) are found in many E. coli strains of the standard ECOR collection (but not in K-12). Inser- tion seems to take place near one terminus of a 121 bp sequence that includes the 3' end of an open reading frame of unknown function.

Perhaps the simplest model for the origin of the 21 phage insertion site is given in Figure 1. We suppose that an ancestor of 21 inserted to the right of icd, and that imprecise excision generated a phage that carried some flanking DNA including part of icd; just as lambda occasionally excises to produce k gal phages (Campbell, 1962). Initially, this new phage inserted and excised by homologous recombination. Later, it developed an integrase (or modified one it already had) to recognize the site it now sees. This provided the advantage assumed to justify the existence of site-specific insertion in lambdoid phages; namely, that the expression of genes for insertion and excision is under phage control. In lambda, the evidence is compelling that their expression is regulated in a teleologically in- telligible manner (Echols & Guarneros, 1983).

Changes in insertion specificity

If 21 were the world's only lambdoid phage, the scheme of Figure 1 might not require further dis- cussion. However, lambdoid phages with various insertion sites all have integrase genes in the same position on their genetic maps, and their integrase genes are clearly related to one another. The inte- grase protein of 21 is identical to that of lambda in 35% of its amino acids. Phage HK022, with a dif- ferent insertion site from lambda but excisionase function interchangeable with lambda's has an inte- grase 74% identical to lambda's. The.integrase of phage 434, which inserts at the same site as lambda, is 99% identical (Campbell, et al., 1990; Baker, et al., 1991; Yagil et al., 1989).

Obviously each of these phages could not have inserted near its present site in the manner indicated in Figure 1 and then evolved an integrase function from scratch. If the model has generality, it forces us to consider the possibility of reprogramming an

Table 1. Heat pulse curing of prophage 21-kHy ci857 a.

Strain Carried Percent Fraction elements b curing for Tc ~ d

prophage c

BS15A (21-XHyc1857 19% 0/100 (el4::Tnl0)

S1731 (21-kHyc1857 <.05% N.D xis::Km) (e 14: :Tn 10) e

a The 21-kHy c1857 phage was constructed by growing phage 21 on strain R978, which is a hbio69 lysogen with a deletion that removes ~B-chlA. Phage that plated on a 21 lysogen were recovered and shown to insert at the 21 site by Southern hybridization with an attB probe. b Lysogens of strain CHl494 (Brody et aL, 1985) which is tetracycline resistant because of a TnlO insertion in el4. c Cells were shifted to 43 ° for 5 rain, then grown at 30 ° for 4 h to allow segregation (Weisberg & Gallant, 1967). Percent curing is the ratio of colony counts at 43 ° and 30 ° . d Fraction of tetracycline sensitive cells among those that had lost the prophage. N.D. = not done.

The Km insert was spliced into the xis gene of an attP segement cloned into a plasmid, then crossed into 21-k Hy CI857.

existing integrase gene rather than creating a new one. The basis for specificity of site recognition is incompletely understood, but certainly changes in specificity of some DNA binding proteins require alteration of only a few critical bases (Wharton & Ptashne, 1985; Gardella et al., 1989). Sequence comparisons of the integrase genes of lambda and HK022 and tests of the activity of each on various combination of sites give some indication of the location of specificity determinants (Yagil et al., 1989).

Lambda and 434 insert at the same site and have the same insertion specificities. What about 21 and el4? No integrase gene has yet been identified in

icd

263

el4, although a relatively weak insertion]excision function is implicated by the promotion of site- specific recombination following UV induction (Brody & Hill, 1988). We have extensive evidence that 21 does not excise el4 efficiently. This is based both on experiments in which integrase and exci- sionase genes of 21 cloned into plasmids are ex- pressed in cells with el4 in the chromosome and on heat-pulse curing of a 21-h recombinant with the lambda ci857 repressor gene in a cell where the 21 and e 14 are adjacent in the chromosome. Phage 21 is efficiently excised from the chromosome when the 21 genes are expressed, but el4 is not (Table 1). Therefore, the sites at the e 14 termini are not recog- nized efficiently by 21 integrase.

In lysogenization, 21 inserts at the left end of el4, not at the right (S. Schneider and A. Campbell, unpublished data). This is consistent with the fail- ure to cure, if the problem is inefficient recognition of the right end of el4 in both cases. Figure 2 depicts a 21 lysogen of a strain carrying e 14, where the termini of the hybrid insertion sites are labeled B, B', P,P', E or E', depending on whether they originated from chromosome, phage 21 or el4. 21 acts on sites with various combinations of B,B', P,P' and E' but not on the EC' site.

Although there is no direct evidence on sequence recognition at the insertion site, we looked for a consensus sequence positioned similarly to lambda's with respect to the crossover point. In lambda, the consensus sequence at the crossover point is the palindrome CAACTTNNTNNNAN NAAGTTG, where the underlined seven bases rep- resent the branch migration region between the cleavage points on the two strands (Weisberg & Landy, 1983). Comparison of the B, B ', P,P ' and E' sequences turns up the consensus sequence CPyNATATGNNGCNGCNNCATATNPuG,

v ~ ~ ~ v

Fig. 2. Structure of a lysogenic chromosome harboring both 21 and e 14. Some restriction sites within the 165 bp homologous Y end of icd are shown. B, P', P, E', E and B', are sequences flanking the crossover points for specific insertions and excisions. BB' is the site (attC) present in the element-free K12 chromosome; PP' is att of phage 21; EE' is attE of el4.

264

which has the same spacing. The E sequence differs from the first half of this sequence at four p)aces.

An important question (not fully answered) is whether el4 has a different specificity from 21 or simply excises poorly because the E sequence is a poorer substrate.

Variation in icd sequence

Phage 21, e14 and the K-12 chromosome have three different 3' ends for the icd gene, diverging from one another in about 7% of their nucleotides. If we assume that the 21 and el4 acquired their icd sequences from the host in a manner similar to that shown in Figure 1, the sequence divergence might arise from several sources: (a) an ancient origin of phage and el4 sequences from the K-12 chromosome, followed by many neutral substitutions (genetic drift) in all three, with little general recombination between the elements and the chromosome. (b) recent derivation of phage and el4 sequences from other enteric hosts that differ from K-12 by this degree. (This could either be a recent origin in such hosts by the mechanism of Figure 1 or later replacement of sequences by homologous recombi- nation with the host). (c) evolutionary pressure for changes that improve the sequences in 21 and el4 as integrase recogni- tion sites. (d) evolutionary pressure to diverge, thereby in- creasing the phage's regulatory control over inser- tion and excision by reducing the rate of unpro- grammed integrations or excisions through general recombination.

The third and fourth possibilities imply that the elements can contribute significantly to the degree of polymorphism of icd genes in the E. coli popula- tion. An even more intriguing notion is that this polymorphism is functionally significant. One could for example imagine that the variation in icd introduced by 21 or el4 might be selectively advan- tageous in special environments, thereby providing selective pressure to maintain these elements in the bacterial population. At the population level, the system generates alternative spliced protein prod- ucts through insertion or excision just as the DNA invertase of phage Mu generates them by inversion. On paper, a nonspecific inversion with one end-

point in prophage DNA and one end point in flank- ing bacterial DNA would convert the phage system into a site specific inversion system. However, a close evolutionary relationship between phage inte- grases and known DNA invertases is untenable, because their biochemistry is substantially different (Craig, 1988).

Possibilities (a) and (d) require that homologous recombination between icd sequences be inconse- quential over evolutionary time. We consider that quite unlikely because, even at their present state of divergence, element and host sequences have sub- stantial opportunities for homologous recombina- tion. We have examined rates of spontaneous pro- phage loss from lysogens of 21-hci857, in some cases carrying mutations in int or xis. The fre- quency of cured cells is typically around 10 -5 . We have no trouble isolating products of excision (both phage and bacterial) at sites within the 165 bp du- plication other than the crossover point, as indi- cated by Southern hybridizations that monitor the restriction sites shown in Figure 2. (B. Song and A. Campbell, unpublished). Possibility (c) remains vi- able and could generate adaptive polymorphisms, but any beneficial variations should soon find their way into the chromosomes of phage-free hosts.

Distribution and orientation of lambdoid proph- ages

Among the known lambdoid prophages, both the distribution of preferred insertion sites and their

HK253 / ~ ~ ~ P 2 2

/ "' 12~DLP12 (qsr')

/ 17~ ~'' 434' 82' 81

/ ~. cotl r..- I z ; 8 ~ 4

\ 30 "J'rac - -~ 080 . . 3 5 / .... Atlas

qm

HK139 Fig. 3. Chromosomal locations of lambdoid prophages.

265

A . i cd ........... - 'ic,.. .....d 21 'i cd

k . . . . . . . ~ ~ ~ . . , ~ " ' ' "

B.

I

icd

\ \ \ \

\!

] icd . ' icd 2 1 icd' icd

C .

i C d ' ~ f l l f f A

i c d -

/) post21 icd

Fig. 4. Possible evolution from phage 21 of a lambdoid phage with a new insertion specificity (by 'chromosomal walking'.) (A) Abnormal excision of a specialized transducing 21 carrying the entire icd gene; - (B) Insertion of 21 icd by homologous recombination; - (C) Insertion of a derivative of this phage by sitespecific recombination to the left of icd. Post 21 differs from the 21 icd of (B) in insertion specificity and in deletion of much of icd.

266

orientation with respect to the E. coli chromosome are non-random. All of the insertion sites lie in one segment of the genetic map - from 6 min to 45 min (Fig. 3). In all but one, the orientation of the pro- phage is the same,

The sample includes phages from several sources. Jacob and Wollman (1961) examined 12 temperature coliphages collected in Paris. Of these, six were related to lambda and inserted in one seg- ment of the chromosome, whereas the other six inserted elsewhere. The K-12 strain harbors, in ad- dition to lambda, four defective lambda-related phages (including el4), all between 12 and 34 min on the K-12 map. Phages such as ~b80 (Japan), HK022 (Hong Kong), P22 (Salmonella phage) and the Atlas prophages from the worldwide ECOR collection all insert between 6 and 28 mins. The diverse sources make it hard to attribute the result to sampling bias.

As to orientation, two of the defective prophages in E. coli K-12 have the same orientation as lambda. One (the qin prophage at 34 min) is oppos- itely oriented, el4 contains no genes observed in lambdoid phages, so its orientation is undeter- mined. HK022, P22, qb80, 21 and the Atlas pro- phages all have the same orientation as lambda. (Dhillon, 1981; Sussldnd & Botstein, 1978; Frank- lin et al., 1965; Stoltzfus, 1991).

The primary cause of the nonrandomness might be either functional or historical. Functional expla- nations have been given for the nonrandom orienta- tion of highly transcribed operons with respect to direction of replication (Brewer, 1990). The exact molecular sequelae of passage of the replication fork over a prophage are unknown. One problem is that the number of operator sites doubles so that some may be transiently underoccupied by re- pressor - which could induce either phage produc- tion or curing in a small fraction of the cells. The lambdoid phages are so oriented that the leftward operon is replicated before the rightward operon, whatever effect that may have. Another possibility in principle is that the proximity of prophage inser- tion sites promotes favorable prophage-prophage interactions (or discourages unfavorable ones). Re- combinational interactions between prophages at different sites certainly can occur (Redfield & Campbell, 1987), although they need not be locus or orientation-dependent (Campbell et al., 1990).

The simplest historical origin for the distribution

of sites is that the E. coli chromosome has a chim- eric origin, the 6-45 min segment having been con- tributed by a host in which lambdoid phages were already established. Explaining the preferred orien- tation requires more imagination. Suppose that Fig- ure 1 represents, not just a model for the way that phage 21 acquired its icd DNA but a more general picture of how phages change location. In the next move, a rare 21 that has picked up the entire icd gene might arise, perhaps enjoy a temporary selec- tive advantage in some special host or environment, then modify its integrase to recognize a site further to the left. Iterated, the process could allow the prophage to 'walk' along the chromosome during evolutionary time, retaining its orientation (Fig. 4).

Both these historical explanations require that existing phages refined their specificities for their present sites far in the past and rarely exploit the potentiality to insert at secondary sites and evolve new specificities. Indeed, the whole 'walking' process becomes more plausible if it occurred at a time when integrases were much less efficient than at present, so that insertion and excision by homol- ogous recombination were more significant. On a more general level, we note that the relative ease with which DNA binding proteins can change spec- ificity by altering their binding domains leaves open the question of how much additional fine tun- ing must follow such a primary change to produce a well-adapted protein; which in turn opens the question of whether most of the specificity varia- tion we now observe may have come about at an earlier time when the ancestral protein was less refined.

We presume at any rate that an important factor in promoting the survival of phages with different insertion specificities has been frequency-depend- ent selection within the phage population. Because infection by a heteroimmune phage with the same insertion specificity can cause curing or substitu- tion of a resident prophage, a rare new type will be able to lysogenize all available hosts and be curable by no other phages and should therefore increase until it becomes common.

Acknowledgement

This research was supported by grant AI08753, National Institute of Allergy and Infectious Dis- eases.

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