The phi 80 and P22 attachment sites. Primary structure and interaction with Escherichia coli integration host factor

Although the lambdoid bacteriophage phi 80 and P22 possess site-specific recombination systems analogous to bacteriophage lambda, they have different attachment (att) site specificities. We have identified and determined the nucleotide sequences of the att sites of phi 80 and P22 and have examined the interaction of these sites with purified Escherichia coli integration host factor (IHF). The sizes of the homologous core regions of the att sites vary greatly: P22 has a 46-base pair core, while phi 80 and lambda have 17- and 15-base pair cores, respectively. The core sequences of the three phage show no significant homology, although dispersed regions of homology in arm sequences indicate that the three phage att sites are related. All three att sites have a high A + T composition, and restriction fragments carrying these sites migrate anomalously upon polyacrylamide gel electrophoresis. IHF binds to a site to the left of the common core in the phi 80 and P22 phage att sites (attP) and to a site to the right of the core in P22 attP and attB (the bacterial att site). In the lambda system, IHF interacts with three regions on attP (designated H1, H2, and H') and none on attB (Craig N., and Nash, H.A. (1984) Cell 39, 707-716). Alignment of the IHF sites of all three phage results in a consensus sequence for IHF binding, Pyr-AANNNNTTGATAT. Among the three phage, the number of IHF sites differs; however, the location and orientation of the binding sites in relation to the respective core regions are well conserved. An IHF site analogous to lambda H2 is present in both phi 80 and P22 attP, while a site analogous to lambda H' is present in P22 attP. This conservation suggests that IHF plays a very similar role in the site-specific recombination pathways of all three phage, and that the flanking arm sequences are necessary for phi 80 and P22 attP function, as is the case for lambda attP function. These structural similarities presumably reflect a conservation of the mechanism of site-specific recombination for the three phage.     

Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU.

The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage lambda. This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min. Immediately clockwise of DLP12 was the IS3 alpha 4 beta 4 insertion element. The argU and DLP12 int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site. Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU. These included the cryptic prophage qsr' (P. J. Highton, Y. Chang, W. R. Marcotte, Jr., and C. A. Schnaitman, J. Bacteriol. 162:256-262, 1985), a sequence homologous to a portion of lambda orf-194, and an attR homolog. Inasmuch as the DLP12 att int xis exo P/ren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences. This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E. coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage. Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages.     

Determinants of site-specific recombination in the lambdoid coliphage HK022. An evolutionary change in specificity

The temperate bacteriophage HK022, like its relative lambda, inserts its chromosome into a specific site in the bacterial chromosome during lysogenization and excises it after induction. However, we find that the recombinational specificities of the two phages differ: they use different bacterial sites, and neither promotes efficient insertion or excision of the other phage chromosome. In order to determine the basis for this difference in specificity, we sequenced the HK022 elements that are involved in insertion and excision, and compared them to the corresponding lambda elements. The location, orientation, size and overall arrangement of the int and xis genes and the phage attachment sites are nearly identical in the two genomes, as is common for other functionally related elements in lambdoid phages. The Xis proteins of the two phages are functionally interchangeable, and their predicted amino acid sequences differ by but one residue. In contrast, the two Int proteins are not functionally interchangeable, and their sequences, although similar, differ at many positions. These sequence differences are not uniformly distributed: the amino-terminal 55 residues are completely conserved, but the remaining 302 show a pattern of differences interspersed with identities and conservative changes. These findings imply that the specificity difference between HK022 and lambda site-specific recombination is a consequence of the inability of the respective Int proteins to recognize pairs of heterologous attachment sites. The two phage attachment sites are remarkably similar, especially the two "arm" segments, which in lambda contain binding sites for Int, Xis and integration host factor. They are less similar in the segment between the two arms, which in lambda contains the points of recombinational strand exchange and a second class of binding site for Int protein (the "core-type" sites). The two bacterial attachment sites are quite different, although both have a short stretch of perfect homology with their respective phage partners at the points of strand exchange. We propose that the two Int proteins recognize similar or identical sites in the arms of their cognate attachment sites, and that differences in binding or action at the core-type sites is responsible for the divergent specificities. Genetic experiments and sequence comparisons suggest that both proteins recognize different but overlapping families of core-type sites, and that divergence in specificity has been achieved by an alternating succession of small, mutually compatible changes in protein and site.     

Some Properties of Site-Specific and General Recombination Inferred from Int-Initiated Exchanges by Bacteriophage Lambda

The site-specific recombination at the attachment site for prophage integration might proceed by two general mechanisms: (1) a concerted reaction without a free intermediate; (2) a sequential mechanism differing from typical general recombination only by an inability of the cross-strand intermediate structure to migrate into the region of nonhomology adjacent to the attachment site. The blocked-migration model predicts frequent genetic exchange in the int xis region near the attachment site if Int-mediated recombination occurs between lambda phage with homologous attachment sites. We find such additional int xis exchanges, but only at very low frequency (1% of the Int-mediated recombination). We conclude that the resolution point only rarely moves away from the initial crossover point specified by Int and, therefore, that the Int reaction is mainly concerted. We interpret the rare additional int xis recombinants as indicative of occasional branch migration from an initial Int-mediated crossover. The frequency of the rare int xis recombinants is not simply related to distance from the attachment site to an int- or xis- mutation, suggesting that the heteroduplex distance is often at least a gene in length. The frequency of these additional exchanges is also not a strong function of distance between two mutations; from this we conclude that the resolution to the observed recombinant structure in the sequential cases occurs often by mismatch repair. We have found no marked effect of mutations in the bacterial recA, recB, recC, recF, or recL genes on the frequency of the int xis recombinants; this may indicate that none of these genes specifies a product uniquely required for resolution of a cross-strand intermediate.     

Sequence analysis and comparison of int and xis genes from staphylococcal bacteriophages L54a and phi 11.

The DNA fragment encoding the integrase and excisionase genes involved in site-specific recombination of staphylococcal bacteriophage phi 11 was cloned and sequenced. The int and xis genes and the recombination site, attP, were highly clustered in a 1.7-kilobase DNA fragment with the gene order attP-int-xis. The int and xis genes were transcribed divergently, with the int gene transcribed toward the attp site and the xis gene transcribed away from the attP site. The deduced Int is a basic protein of 348 residues with an estimated molecular weight of 41,357. In contrast, the deduced Xis is an acidic protein containing 66 amino acids with an estimated molecular weight of 7,621. The site-specific recombination system of phi 11 was compared with that of a closely related bacteriophage, L54a.     

Structural analysis of the actinophage phi C31 attachment site.

The lysogenisation of actinophage phi C31 in S. coelicolor J 1501 occurs by site-specific recombination. The DNA segments containing the attachment sites on the host chromosome, the phage genome and the two junctions created by the insertion of the prophage were cloned and the nucleotide sequences determined. The attachment sites (att) share an extremely short common sequence of three base pairs. Adjacent to the core sequences some direct- and inverted repeats were found as potential binding sites for proteins involved in site-specific recombination.     

Identification of Site-Specific Recombination Genes int and xis of the Rhizobium Temperate Phage 16-3

Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis (prophage excision). We concluded that Int protein of phage 16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.     

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.     

Structural analysis of staphylococcal bacteriophage phi 11 attachment sites.

The lysogenization of bacteriophage phi 11 in Staphylococcus aureus occurs by site-specific recombination. The DNA segments containing the attachment sites on the host chromosome, the phage genome, and the two junctions created by insertion of the prophage were cloned, and the nucleotide sequences were determined. The attachment sites share a very short common sequence of 10 base pairs.     

Localization and nucleotide sequences of genes mediating site-specific recombination of the SLP1 element in Streptomyces lividans.

SLP1 is a 17.2-kbp genetic element indigenous to the Streptomyces coelicolor chromosome. During conjugation, SLP1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells. We report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events. A region of SLP1 adjacent to the previously identified site of integration, attP, was found to be sufficient to promote site-specific integration of an unrelated Streptomyces plasmid. Nucleotide sequence analysis of a 2.2-kb segment of this region reveals two open reading frames that are adjacent to and transcribed toward the attP site. One of these, the 1,365-bp int gene of SLP1, encodes a predicted 50.6-kDa basic protein having substantial amino acid sequence similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage lambda integrase. A linker insertion in the 5' end of the cloned int gene prevents integration, indicating that Int is essential for promoting integration. An open reading frame (orf61) lying immediately 5' to int encodes a predicted 7.1-kDa basic peptide showing limited sequence similarity to the excisionase (xis) genes of other site-specific recombination systems.     

The integrase family of site-specific recombinases: regional similarities and global diversity.

A combination of two methods for detecting distant relationships in protein primary sequences was used to compare the site-specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1. This group of proteins exhibits an unexpectedly large diversity of sequences. Despite this diversity, all of the recombinases can be aligned in their C-terminal halves. A 40-residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein. This family of recombinases does not appear to be related to any other site-specific recombinases. Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well-conserved C-terminal region. We speculate that these residues contribute to the active site of this family of recombinases, and suggest that tyrosine-433 forms a transient covalent linkage to DNA during strand cleavage and rejoining.     

Nucleotide sequence and genetic characterization of staphylococcal bacteriophage L54a int and xis genes.

The nucleotide sequence of a staphylococcal bacteriophage L54a DNA fragment containing genes involved in site-specific recombination was determined. Mutations generated by in vitro mutagenesis were used to map and characterize the int and xis genes. The site-specific recombination functions are tightly clustered within a 1.75-kilobase stretch of DNA fragment with the gene order of attP-int-xis. The int and xis genes are transcribed divergently. The Int protein deduced from the nucleotide sequence has a molecular weight of 41,000. Int is a basic protein with 354 amino acids of which 72 are basic and 38 are acidic. The Xis protein consists of only 59 amino acids with a molecular weight of 7,180. Unlike the Xis proteins of the lambdoid bacteriophages which are all basic proteins, L54a Xis is an acidic protein containing 13 acidic and 8 basic amino acids. The Int protein is required in both integrative and excisive reactions, whereas Xis is only required in excisive reaction. A well-conserved 40-residue region, including three perfectly conserved residues found in 15 site-specific recombinases of the integrase family that have been characterized, was also found in the L54a Int protein.     

Analysis of insertion into secondary attachment sites by phage lambda and by int mutants with altered recombination specificity

When phage lambda lysogenizes a cell that lacks the primary bacterial attachment site, integrase catalyzes insertion of the phage chromosome into one of many secondary sites. Here, we characterize the secondary sites that are preferred by wild-type lambda and by lambda int mutants with altered insertion specificity. The sequences of these secondary sites resembled that of the primary site: they contained two imperfect inverted repeats flanking a short spacer. The imperfect inverted repeats of the primary site bind integrase, while the 7 bp spacer, or overlap region, swaps strands with a complementary sequence in the phage attachment site during recombination. We found substantial sequence conservation in the imperfect inverted repeats of secondary sites, and nearly perfect conservation in the leftmost three bases of the overlap region. By contrast, the rightmost bases of the overlap region were much more variable. A phage with an altered overlap region preferred to insert into secondary sites with the corresponding bases. We suggest that this difference between the left and right segments is a result of the defined order of strand exchanges during integrase-promoted recombination. This suggestion accounts for the unexpected segregation pattern of the overlap region observed after insertion into several secondary sites. Some of the altered specificity int mutants differed from wild-type in secondary site preference, but we were unable to identify simple sequence motifs that account for these differences. We propose that insertion into secondary sites is a step in the evolutionary change of phage insertion specificity and present a model of how this might occur.     

Lambda H-lambda T 80 hybrid study of the DNA structural gene region in lambda and phi 80 phages

Hybrids lambda H lambda T80 are formed due to recombination of the phage lambda att80 and phi 80 prophage partially deleted in the region of structural genes. Genetic structure of 22 independently isolated lambda H lambda T80 hybrids was determined by the restriction method and it was shown that recombination took place in the genes A, C, D and H. The frequencies of hybrid formation diminish from 1.10(-3) to 4.10(-5) for this gene order, which suggests that the polar divergence of nucleotide sequencies in the region of structural genes exists. It was found that formation of hybrids with recombination in the region of "weak" homology (gene H) was possible only when the region of "strong" homology was present in the deleted phi 80 prophage to initiate recombination.     

Escherichia coli plasmid vectors for high-level regulated expression of the bacteriophage lambda xis gene product

The bacteriophage lambda Xis protein is one of the proteins required for site-specific excisive recombination by which the lambda prophage is excised from the Escherichia coli bacterial chromosome. We cloned the lambda xis gene under the control of several prokaryotic promoters to obtain a sufficient source of the protein for biochemical studies. Our results demonstrate that E. coli lac promoter and lambda pL promoter fusions to the xis gene produce high levels of Xis protein. Induction of the expression vectors results in a 10- to 50-fold increase in Xis activity. In addition, one of these plasmids allows the control of xis expression in vivo.