Summary Hin, a member of the serine family of site-specific recombinases, regulates gene expression by inverting a DNA segment. DNA inversion requires assembly of an invertasome complex in which a recombinational enhancer DNA segment bound by the Fis protein associates with the Hin synaptic complex at the base of a supercoiled DNA branch. Each of the four Hin subunits becomes covalently joined to the cleaved DNA ends, and DNA exchange occurs by translocation of a Hin subunit pair within the tetramer. We show here that although the Hin tetramer forms a bidirectional molecular swivel, the Fis/enhancer system determines both the direction and number of subunit rotations. The chirality of supercoiling directs rotational direction, and the short DNA loop stabilized by Fis-Hin contacts limit rotational processivity, thereby ensuring that the DNA strands re-ligate in the recombinant configuration. We identify multiple rotational conformers that are formed under different supercoiling and solution conditions.

Many surface proteins of Gram-positive bacteria are anchored to the cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. In Staphylococcus aureus, surface proteins are cleaved between the threonine and the glycine of the LPXTG motif. The carboxyl of threonine is subsequently amide linked to the amino group of the pentaglycine cell wall crossbridge. Here we investigated the anchor structure of surface proteins in Listeria monocytogenes. A methionine and six histidines (MH(6)) were inserted upstream of the LPXTG motif of internalin A (InlA), a cell-wall-anchored surface protein of L. monocytogenes. The engineered protein InlA-MH(6)-Cws was found anchored in the bacterial cell wall. After peptidoglycan digestion with phage endolysin, InlA-MH(6)-Cws was purified by affinity chromatography. COOH-terminal peptides of InlA-MH(6)-Cws were obtained by cyanogen bromide cleavage followed by purification on a nickel-nitriloacetic acid column. Analysis of COOH-terminal peptides with Edman degradation and mass spectrometry revealed an amide linkage between the threonine of the cleaved LPXTG motif and the amino group of the m-diaminopimelic acid crossbridge within the listerial peptidoglycan. These results reveal that the cell wall anchoring of surface proteins in Gram-positive bacteria such as S. aureus and L. monocytogenes occurs by a universal mechanism.

BackgroundA large subfamily of serine recombinases contains long polypeptide segments appended to the C-terminal end of the conserved catalytic domain. Members of this subfamily often function as phage integrases but also mediate transposition and regulate terminal differentiation processes in eubacteria. Although a few members of this subfamily have been studied in purified in vitro systems, key mechanistic aspects of reactions promoted by these recombinases remain to be determined, particularly with respect to the functions of the large C-terminal domain.ResultsWe have developed and characterized a robust in vitro recombination reaction by the Listeria phage A118 integrase, a member of the subfamily of serine recombinases containing a large C-terminal domain. The reaction occurs in a simple buffered salt solution and exhibits a modest stimulation by divalent cations or spermidine and DNA supercoiling. Recombination with purified A118 integrase is unidirectional, being efficient only between attP and attB DNA sites to either join separate DNA molecules (intermolecular recombination) or to generate deletions or inversions depending on the relative orientation of att sites in cis (intramolecular recombination). The minimal attP site is 50 bp but requires only 44 bp of base sequence information, whereas the minimal attB site is 42 bp and requires 38 bp of base sequence information. DNA exchange occurs between the central 2 bp of attP and attB. Identity between these two base pairs is required for recombination, and they solely determine the orientation of recombination sites. The integrase dimer binds efficiently to full att sites, including the attL and attR integration products, but poorly and differentially to each half-site. The large C-terminal domain can be separated from the N-terminal catalytic by partial proteolysis and mediates non-cooperative DNA binding to att sites.ConclusionsThe basic properties of the phage A118 integrase reaction and its substrate requirements have been elucidated. A118 integrase thus joins the handful of biochemically characterized serine integrases that are serving as models for mechanistic studies on this important class of recombinases. Information reported here will also be useful in exploiting this recombinase for genetic engineering.

Most site-specific recombinases can be grouped into two structurally and mechanistically different classes. Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements. Here we use site-directed crosslinking to investigate the conformational changes that accompany the formation of the synaptic complex and the exchange of DNA strands by the Hin serine recombinase. Efficient crosslinking between residues corresponding to the ‘D-helix’ region provides the first experimental evidence for interactions between synapsed subunits within this region and distinguishes between different tetrameric conformers that have been observed in crystal structures of related serine recombinases. Crosslinking profiles between cysteines introduced over the 35 residue E-helix region that constitutes most of the proposed rotating interface both support the long helical structure of the region and provide strong experimental support for a subunit rotation mechanism that mediates DNA exchange.

Most site-specific recombinases can be grouped into two mechanistically distinct families. Whereas tyrosine recombinases exchange DNA strands through a Holliday intermediate, serine recombinases such as Hin generate double-strand breaks in each recombining partner. Here, site-directed protein crosslinking is used to elucidate the configuration of protein subunits and DNA within the Hin synaptic complex and to follow the movement of protein subunits during DNA strand exchange. Our results show that the protein interface mediating synapsis is localized to a region within the catalytic domains, thereby positioning the DNA strands on the outside of the Hin tetrameric complex. Unexpected crosslinks between residues within the dimerization helices provide evidence for a conformational change that accompanies DNA cleavage. We demonstrate that the Hin subunits, which are linked to the cleaved DNA ends by serine-phosphodiester bonds, translocate between synapsed dimers to exchange the DNA strands.