Conservation of xer site‐specific recombination genes in bacteria

A major role of homologous recombination is to allow stalled or broken replication forks to reform and thereby re-initiate replication (see Cox, 1998, Genes Cells 3: 65– 78). In a circular chromosome, a likely consequence of this recombinational repair is the generation of dimeric chromosomes that must be resolved to monomers prior to segregation to daughter cells at cell division. In Escherichia coli, chromosomal dimers are converted to monomers by Xer site-specific recombination. Two related recombinases, XerC and XerD, act a specific site, dif, located in the replication terminus region of the chromosome (see Sherratt et al., 1995, Phil Trans R Soc Lond 347: 37– 42). Xer-mediated dimer resolution in E. coli is co-ordinated with other cellular processes, so that Xer recombination at dif occurs only under conditions that allow chromosome dimer formation and in which the C-terminal domain of the cell division-related protein, FtsK, is present (Steiner and Kuempel, 1998, J Bacteriol 180: 6269–6275; Recchia et al., 1999, EMBO J, 18: 5724–5734; Steiner et al., 1999, Mol Microbiol 31: 579–583). Apparent homologues of XerC and XerD have been identified in a variety of Gramnegative and Gram-positive bacteria (Esposito and Scocca, 1997, Nucleic Acids Res 25: 3605–3614; Nunes-Duby et al., 1998, Nucleic Acids Res 26: 391–406). XerC and XerD belong to the large tyrosine recombinase family, members of which are highly divergent in sequence, and function in many different aspects of DNA metabolism. Alignments of tyrosine recombinase sequences reveal that Xer-like proteins form a distinct subfamily (Esposito and Scocca, 1997, ibid). Do all bacteria with circular chromosomes utilize Xer recombinases to resolve dimers? How ancient is the Xer recombination system? The accumulation of complete microbial genome sequences has made it possible to begin to address these questions. We have analysed the complete genomes of 16 eubacteria and five archaea (http:/ /www.tigr.org) for the presence of Xer homologues. Initially genome databases were searched using the most highly conserved C-terminal catalytic domains (<190 aa) of the E. coli and Bacillus subtilis Xer recombinases. As related sequences were identified, these were in turn used to search other genome databases. A compilation of the identified Xer homologues is presented in Table 1. Identity between the sequences in Table 1 ranges from 23% to 69% (45–82% similarity). The majority of eubacteria possess two putative Xer recombinases, suggesting that the mechanism of dimer resolution used by E. coli is highly conserved. Most eubacteria for which only partial genome sequences are available also possess two Xer proteins, including Pseudomonas, Vibrio, Bordatella, Neisseria, Staphylococcus and Enterococcus species (G. D. Recchia, G. Blakely and D. J. Sherratt, unpublished). To date, dif sites have only been found in E. coli, Salmonella typhimurium and Haemophilus influenza ; in each case they are located in the replication terminus region. Although the two Xer homologues within a species are often more closely related to each other than to the Xer homologues of other species, we believe it likely that XerC and XerD evolved from a single ancestral protein via a gene duplication event and that these separate genes have been transmitted through bacterial lineages. Relatedness analyses do not allow us to distinguish this phylogenetic origin from processes in which gene duplication occurred frequently in different evolutionary branches. It is likely that functional constraints prevent xerC and xerD genes from diverging substantially in a species, as intricate interactions between XerC and XerD are needed to assemble a recombination complex and to mediate coordinated catalysis. Two eubacterial species appear to contain only one Xer homologue. In these cases, either one Xer protein has been lost or, assuming that xerC and xerD genes arose from a single ancestral gene, these organisms diverged from other bacterial lineages prior to this duplication. Likewise, organisms in which no Xer-like sequences were identified may have either lost both sequences or separated from other bacterial lineages prior to the evolution of Xer. The spirocheaete Borrelia burgdorferi possesses a linear chromosome and thus has no requirement for chromosome dimer resolution; this organism lacks any identifiable Xer homologues. The apparent absence of Xer homologues in Mycoplasma genitalium and Mycoplasma pneumoniae correlates with their deficiency in homologous recombination genes. Of the more than a dozen homologous recombination genes described in other bacteria, only recA, ssb, ruvA and ruvB are present in M. genitalium and M. pneumoniae (Himmelreich et al., 1997, Nucleic Acids Res 25: 701–712; G. D. Recchia, unpublished). Although it is possible that these organisms utilize an alternative mechanism Molecular Microbiology (1999) 34(5), 1146–1148