Conservation of <i>xer</i> site‐specific recombination genes in bacteria (1999)

Sir, 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 Cells3: 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 Lond347: 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 Bacteriol180: 6269–6275; Recchia et al., 1999, EMBO J, 18: 5724–5734; Steiner et al., 1999, Mol Microbiol31: 579–583). Apparent homologues of XerC and XerD have been identified in a variety of Gram-negative and Gram-positive bacteria (Esposito and Scocca, 1997, Nucleic Acids Res25: 3605–3614; Nunes-Duby et al., 1998, Nucleic Acids Res26: 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 co-ordinated 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 Res25: 701–712; G. D. Recchia, unpublished). Although it is possible that these organisms utilize an alternative mechanism of resolving chromosome dimers, unrelated to Xer recombination, this is unlikely given the otherwise widespread conservation of Xer proteins over long evolutionary distances. Rather, we propose that reductive evolution of these genomes has resulted in the loss of Xer recombinases. It is well documented that in parasitic organsims, including the mycoplasma, reductive evolution has been responsible for the loss of a significant number of metabolic genes (Andersson and Kurland, 1998, Trends Microbiol6: 263–268). It is tempting to suggest that the apparent deficiencies in homologous recombinational capabilities in these species are the result of reductive evolution and that this in turn has led to the loss of Xer recombinases. In contrast, the genomes of the parasitic organisms Rickettsia prowazeckii, Treponema pallidum and Chlamydia trachomatis, which have also been subjected to considerable genomic reduction, each retain a large complement of homologous recombination genes and also possess two Xer homologues. These correlations are consistent with the functional inter-relationship between homologous recombination and Xer recombination. Like most eubacteria, archaea possess circular genomes. As archaea also appear to be proficient in homologous recombination, they presumably possess some mechanism for the resolution of dimers. Sequence similarity analyses suggest that, in general, DNA replication and recombination proteins resemble those found in eukaryotes, whereas putative chromosome partitioning proteins show similarity to both eukaryotic and eubacterial proteins (Bernander, 1998, Mol Microbiol29: 955–961). Of five archaeal species for which complete genome sequences are available, three possess ORFs similar to eubacterial Xer proteins, with each appearing to possess only a single Xer recombinase, whereas in the remaining two species sequences related to Xer proteins could not be identified. The level of identity shared by the Xer recombinases in Methanobacterium thermoautotrophicum, Pyrococcus horikoshii and Aeropyrum pernix with those of eubacteria (e.g. 32–37% identity with E. coli Xer proteins) is higher than that shared by many eubacterial Xer proteins, and may suggest that these archaeal homologues evolved by horizontal gene transfer of a eubacterial enzyme(s) into the archaea. It will be interesting to determine if these archaeal Xer-like proteins play a role in chromosome segregation similar to that of the eubacterial recombinases or have some other function. Similarly, it will be interesting to determine how the problem of dimers is solved in circular organelle genomes. Despite the need to express both XerC and XerD for recombination to occur, an intriguing aspect of those genomes that possess two xer genes is that these genes are separated on the chromosome with no evidence for common controlling elements. With the exception of T. pallidum, the xerC and xerD genes are distant from each other on the chromosome and, in all but one of these species, are divergently transcribed with respect to each other (see Table 1). This contrasts with the situation in which genes encoding products of related function are often located in operons, in close proximity on the chromosome, or at least co-ordinately regulated when distantly located. This is true not only of metabolic genes but also of genes involved in DNA replication and recombination, e.g. recF/dnaA/dnaN, ruv genes, recBC (and often recD ) and sbcCD. Furthermore, in one of the few other examples of a site-specific recombination system employing two recombinases, FimB and FimE, which control fimbrial gene expression in E. coli, the genes are located adjacent to one another (Klemm, 1986, EMBO J5: 1389–1393). Xer-mediated chromosome dimer resolution is part of an integrated network of events that link DNA replication, homologous recombination, chromosome segregation and cell division. At present, we understand only a few of the links in this network. The genomic separation of xerC and xerD, and the fact that each is often co-expressed with other genes, some of which are implicated in these networks, may be of some functional significance. Anecdotally, we note that genes implicated directly or indirectly in recombination are often located close to one or both xer genes, for example in E. coli, xerD is in the same operon as recJ, whereas xerC is proximal to the uvrD and recQ genes, in H. influenzae, xerD is adjacent to the ruvABC operon, and in M. tuberculosis, the xerD gene is close to recN. Xer recombination at dif requires the C-terminus of the septum-located protein, FtsK (Steiner et al., 1999, ibid; Recchia et al., 1999, ibid). Analysis of complete genome sequences reveals that FtsK is widely conserved in eubacteria, but appears to be absent in the archaea (Table 1). There appears to be a correlation between the presence of Xer and FtsK homologues in eubacterial species; all species possessing Xer homologues also encode an FtsK-like protein, whereas Aquifex aeolicus, M. genitalium and M. pneumoniae, which lack identifiable Xer homologues, also appear to lack an FtsK homologue. This suggests that the functional interaction between Xer and FtsK proteins in controlling chromosome dimer resolution is highly conserved, and may provide a further functional constraint on xer gene evolution. This analysis reveals that the Xer site-specific recombination system is a highly conserved mechanism for the maintenance of genome integrity in bacteria. Additionally, the observations reported here, in particular the physical separation of xer genes, the presence of archaeal Xer homologues and their apparent absence from several eubacterial genera raise interesting questions for future research. G.D.R. has been supported by a C. J. Martin Postdoctoral Fellowship from the NHMRC of Australia and the Wellcome Trust. D.J.S. is supported by the Wellcome Trust.