Specificity determinants for bacteriophage Hong Kong 022 integrase: analysis of mutants with relaxed core-binding specificities

The integrase (Int) proteins encoded by bacteriophages HK022 and lambda catalyse similar site-specific integration and excision reactions between specific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and lambda are unable to catalyse recombination between non-cognate att sites. The att sites of both phages contain weak binding sites for Int, known as 'core-type' sites. Negatively acting nucleotide determinants associated with specific core sites (lambda B', HK022 B', HK022 C) are responsible for the barrier to non-cognate recombination. In this study, we used challenge phages to demonstrate that the lambda and HK022 Ints cannot bind to core sites containing non-cognate specificity determinants in vivo. We isolated mutants of the HK022 Int, which bind the lambda B' core site. Two mutants, D99N and D99A, have changed a residue in the core-binding (CB) domain, which may be directly contacting the core site DNA. We suggest that binding to the lambda B' site was accomplished by removing the negatively charged aspartate residue, which normally participates in a conflicting interaction with the G4 nucleotide of the lambda B' site. We showed that, although our mutants retain the ability to recombine their cognate att sites, they are unable to recombine lambda att sites.     

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.     

Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex

We have determined the genomic sequences of four virulent myophages, Bcep1, Bcep43, BcepB1A, and Bcep781, whose hosts are soil isolates of the Burkholderia cepacia complex. Despite temporal and spatial separations between initial isolations, three of the phages (Bcep1, Bcep43, and Bcep781, designated the Bcep781 group) exhibit 87% to 99% sequence identity to one another and most coding region differences are due to synonymous nucleotide substitutions, a hallmark of neutral genetic drift. Phage BcepB1A has a very different genome organization but is clearly a mosaic with respect to many of the genes of the Bcep781 group, as is a defective prophage element in Photorhabdus luminescens. Functions were assigned to 27 out of 71 predicted genes of Bcep1 despite extreme sequence divergence. Using a lambda repressor fusion technique, 10 Bcep781-encoded proteins were identified for their ability to support homotypic interactions. While head and tail morphogenesis genes have retained canonical gene order despite extreme sequence divergence, genes involved in DNA metabolism and host lysis are not organized as in other phages. This unusual genome arrangement may contribute to the ability of the Bcep781-like phages to maintain a unified genomic type. However, the Bcep781 group phages can also engage in lateral gene transfer events with otherwise unrelated phages, a process that contributes to the broader-scale genomic mosaicism prevalent among the tailed phages.     

DNA sequence of the att region of coliphage 434

Phages lambda and 434 are related phages that insert at the same site on the Escherichia coli chromosome. A 5.9-kb SalI-BamHI fragment derived from phage 434 was shown to hybridize to a 0.5-kb probe carrying attP-lambda. A 0.8-kb Bam HI-TaqI fragment subcloned into pBR327 was used for sequencing. The sequence of the 500 bp around the insertion site is given here, Comparison of the lambda and 434 sequence shows that the following regions are conserved: the coding sequence for the integrase protein (only 162 bp have been sequenced corresponding to the carboxy terminus), the 15-bp common core at the insertion site, and the three integrase-binding sites flanking the insertion site. The lambda and 434 sequences diverge radically to the left of base-197, suggesting that DNA to the left of that point plays no specific role in insertion or its regulation.     

Specificity in DNA recognition by phage integrases

The λ-related (lambdoid) coliphages are related to one another by frequent natural recombination and maintain a high level of functional polymorphism for several activities of the phages. Arguments are presented that the polymorphism of the integration module results from selection (presumably frequency-dependent) for new (not improved) specificities of site recognition. Analysis of phages λ and HK022 by Weisberg and collaborators previously showed that changes in five noncontiguous amino acids could switch site recognition specificity. Phage 21 and defective element e14, which integrate at the same site, differ in recognition specificity for both core and arm sites. In vitro assays of e14 and 21 insertion and excision confirm this conclusion. Inhibition by ds arm site oligonucleotides defines the sequence specificity more precisely.     

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.     

Specificity determinants in the attachment sites of bacteriophages HK022 and lambda.

The Int proteins of bacteriophages HK022 and lambda promote recombination between phage and bacterial attachment sites. Although the proteins and attachment sites of the two phages are similar, neither protein promotes efficient recombination between the pair of attachment sites used by the other phage. To analyze this difference in specificity, we constructed and characterized chimeric attachment sites in which segments of one site were replaced with corresponding segments of the other. Most such chimeras recombined with appropriate partner sites in vivo and in vitro, and their differential responses to the Int proteins of the two phages allowed us to locate determinants of the specificity difference in the bacterial attachment sites and a central segment of the phage attachment sites. The location of these determinants encompasses three of the four core-type binding sites for lambda Int: C, B, and most importantly, B'. The regions corresponding to the C' core binding site and the arm-type binding sites of lambda Int play no role in the specificity difference and, indeed, are well conserved in the two phages. We found, unexpectedly, that the effect of replacement of an Int-binding region on the recombinational potency of one chimeric site was reversed by a change of partner. This novel context effect suggests that postsynaptic interactions affect the specificity of recognition of attachment sites by Int.     

Sequence analysis of Stx2-converting phage VT2-Sa shows a great divergence in early regulation and replication regions.

In enterohemorrhagic Escherichia coli, Shiga toxin is produced by lysogenic prophages. We have isolated the prophage VT2-Sa that is responsible for production of Shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage DNA. The entire DNA sequence consisted of 60,942 bp, exhibiting marked similarity to the 933W phage genome. However, several differences were observed in the immunity and replication regions, where cI, cII, cIII, N, cro, O, and P genes were present: Predicted amino acid sequences of N, cI, cro, O and P in the VT2-Sa genome did not show significant similarity to the counterparts of the 933W genome; however its cI showed higher similarity to lambda. Furthermore, O and P closely resembled those of phage HK022. These observations suggest that the various degrees of homology observed in the immunity and replication regions of VT2-Sa could have resulted from frequent recombination events among the lambdoid phages, and that these regions play a key role as a functional unit for phage propagation in competition with other lambdoid phages.     

Sites and gene products involved in lambdoid phage DNA packaging.

21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage. The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence. The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific. DNA packaging by 21 is dependent on a host protein, integration host factor. A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1. gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase. orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI. orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth. 21 was also not able to complement a lambda FI mutant.     

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.     

A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity

Comparison of eight lambdoid phages, including three Shiga-toxin converting phages, has been carried out with respect to the immunity region, especially the recognition helices of their repressor and CRO proteins on the one hand, and operator sequences on the other. Some as yet unassigned components of the regulatory circuits have been inferred by computer search. The cross immunity phenomenon shown by phages VT2-Sa and lambda is explained on the basis of similarity in their sequences. In addition, the similarity of 933W and HK022 in the sequences of their recognition helices of repressor and CRO, on the one hand, and operators, on the other, has led us to predict that they will have identical or similar immunity specificity. This homology has enabled us also to locate the OL (and consequently PL) of phage 933W that has been thought to be non-existent.