The Bxb1 gp47 recombination directionality factor is required not only for prophage excision, but also for phage DNA replication

Abstract

Mycobacteriophage Bxb1 encodes a serine-integrase that catalyzes both integrative and excisive site-specific recombination. However, excision requires a second phage-encoded protein, gp47, which serves as a recombination directionality factor (RDF). The viability of a Bxb1 mutant containing an S153A substitution in gp47 that eliminates the RDF activity of Bxb1 gp47 shows that excision is not required for Bxb1 lytic growth. However, the inability to construct a Δ47 deletion mutant of Bxb1 suggests that gp47 provides a second function that is required for lytic growth, although the possibility of an essential cis-acting site cannot be excluded. Characterization of a mutant prophage of mycobacteriophage L5 in which gene 54 – a homologue of Bxb1 gene 47 – is deleted shows that it also is defective in induced lytic growth, and exhibits a strong defect in DNA replication. Bxb1 gp47 and its relatives are also unusual in containing conserved motifs associated with a phosphoesterase function, although we have not been able to show robust phosphoesterase activity of the proteins, and amino acid substitutions with the conserved motifs do not interfere with RDF activity. We therefore propose that Bxb1 gp47 and its relatives provide an important function in phage DNA replication that has been co-opted by the integration machinery of the serine-integrases to control the directionality of recombination.

Introduction

Mycobacteriophage Bxb1 is a temperate phage that infects and forms lysogens of Mycobacterium smegmatis, a nonpathogenic relative of Mycobacterium tuberculosis. Bxb1 is one of a large collection of over 80 completely sequenced mycobacteriophage genomes encompassing a large span of genetic diversity (Pope et al., 2011). Although eleven of these encode large serine recombinases, Bxb1 is the only one of the mycobacteriophage serine integrases to be investigated in depth (Ghosh et al., 2003, Ghosh et al., 2005, Ghosh et al., 2006, Ghosh et al., 2008, Kim et al., 2003), along with that encoded by the M. tuberculosis prophage-like element, ϕRv1(Bibb and Hatfull, 2002, Bibb et al., 2005).

The phage-encoded large serine-integrases are of interest because they function catalytically like other serine-recombinases – with concerted cleavage of both DNA strands and covalent linkage to an active site serine residue (Ghosh et al., 2003, Grindley et al., 2006, Smith and Thorpe, 2002) – but without requirement for DNA supercoiling, accessory protein binding sites, or secondary host factors. The attP and attB sites are relatively simple – typically less than 50 bp – with the crossover site centrally located and flanked by imperfect inverted repeats. For mycobacteriophage Bxb1, regulation of directionality is strongly regulated and is dependent on a second phage-encoded protein, Bxb1 gp47 (Ghosh et al., 2006). In its absence, integration proceeds efficiently with just Bxb1 integrase (gpInt) and short DNA substrates containing attP and attB (Ghosh et al., 2003), but no excision is observed. If Bxb1 gp47 is present, then integrative recombination is inhibited and gpInt mediates excisive recombination between attL and attR.

Bxb1 gp47 is an unusual recombination directionality factor (RDF) in that it is unrelated to Xis proteins that work with tyrosine-integrases (Lewis and Hatfull, 2001) and does not bind by itself directly to attachment site DNA, but associates with integrase-DNA complexes (Ghosh et al., 2006). Interestingly, directionality control of Streptomyces phage ϕC31 integrase is determined similarly, although the phage-encoded protein, gp3 is evolutionarily unrelated to Bxb1 gp47 (Khaleel et al., 2011). RDF proteins have also been described for the ϕRv1 and TP901 serine-integrases (Bibb et al., 2005, Breuner et al., 1999) although these are small basic proteins, and related to the Xis proteins usually associated with tyrosine integrases. DNA binding is not required for Bxb1 gp47, ϕC31 gp3, or the ϕRv1RDF (Bibb et al., 2005, Ghosh et al., 2006, Khaleel et al., 2011), and although they have distinct evolutionary origins, they may act similarly to modulate the configuration of integrase-DNA complexes such as to modulate substrate-dependent protein-mediated synapsis.

Seventeen of the currently sequenced mycobacteriophages encode relatives of Bxb1 gp47 (Fig. 1), but the locations, structures, and distributions among the different genes are unusual. First, in Bxb1, gene 47 is not located proximal to the attP site and integrase gene, but displaced approximately 5 kbp to their right, among genes that have putative roles in DNA replication, including a DNA Polymerase and a DNA Primase; it is similarly located in all other ten mycobacteriophages that encode a serine recombinase. Secondly, homologues of Bxb1 gp47 are present in six mycobacteriophages that do not encode a serine-integrase, all of which encode a tyrosine-integrase. The site-specific recombination system of one of these – L5 – has been studied extensively, and all of the components required for efficient integrative and excisive recombination have been identified (Lee and Hatfull, 1993, Lewis and Hatfull, 2000); the L5 gp54 homologue of Bxb1 gp47 is not among them. Lastly, three of the 17 homologues of Bxb1 gp47 – all within serine-integrase encoding genomes, U2, Bethlehem and KBG – contain inteins. Inteins are typically associated with genes required for essential functions (Cheriyan and Perler, 2009), and prophage excision is not typically required for lytic phage propagation of infecting particles. This raises the question as to whether Bxb1 gp47 and its relatives are supporting some additional phage requirement, perhaps in DNA replication during lytic growth.

Here we present evidence supporting a role for Bxb1 gp47 and its homologues in mycobacteriophage DNA replication, in addition to its RDF function. We show that the excision function of Bxb1 gp47 is not required for lytic growth, but that gp47 is likely essential for plaque formation. Furthermore, an L5 prophage in which its gp47 homologue is deleted is unable to undergo efficient lytic induction and exhibits a strong defect in DNA replication. Curiously, these unusual RDF proteins contain motifs associated with phosphoesterase (e.g. phosphatase) activity, although neither Bxb1 gp47 nor its L5 gp54 homologue shows robust phosphoesterase activity in vitro, and substitutions in putative metal coordinating residues do not interfere with excision activity. We propose that Bxb1 and its relatives act similarly in DNA replication and in recombination directionality control, by influencing the active conformations of protein-DNA complexes.