BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/8 These include DNA methyltransferases and the primo- somal protein DnaD from low-GC Gram-positive bacteria [37] that co-occur with both ERF and RecT superfamily members (Figs. 2,4). The poorly characterized phage-or prophage-specific genes that are frequently observed in these neighborhoods include ORF15 (Streptococcus ther- mophilus bacteriophage 7201), ORF86, ORF100a (Staphy- lococcus aureus temperate phage φSLT) and ORF364 (bacteriophage φ31.1) (Figs. 2,4). Secondary structure predictions indicate a high α-helical content for these pro- teins. It is likely that these α-helical proteins are phage in- novations that could function as adaptors in the recombination pathway either as accessory protein-pro- tein interacting domains or as DNA-binding domains. Evidence for convergent operon evolution and in situ non- orthologous displacement of genes in operons encoding SSAPs A superposition of the gene neighborhood information upon the phylogenetic trees for the SSAP superfamilies provides insights into the evolutionary processes that led to the emergence of the operons that include the SSAP genes. As discussed above, the RecT/Redβ superfamily clearly splits into three distinct families (Fig. 2). The phy- logenetic tree shows that SbcC co-occurs with the SSAP once within Redβ-family and once within the RecT-fami- ly. An examination of the tree and the respective gene neighborhoods suggests that independent juxtaposition of SbcC with Redβ-like and RecT-like genes on two sepa- rate occasions is the most parsimonious explanation. The alternative explanation, namely that the gene coding for the common ancestor of the Redβ and RecT already co-oc- curred with the sbcC gene is far less likely because it would require over 10 independent losses of this, apparently, functionally advantageous organization in different bacte- rial and bacteriophage lineages. Likewise, the observation that, in one or more cases, genes encoding each of the SS- APs co-occur in the same predicted operon with SSB or a λ-type exonuclease, suggests that similar operon struc- tures may also emerge independently in evolution. Thus, the same or analogous operon organizations may emerge convergently on multiple occasions, probably due to the selective pressure arising from the strong interactions be- tween the SSAPs and their functional partners such as Sb- cC, SSB and LE. The distribution of the gene neighborhoods, in which a member of the RecT superfamily occurs next to the RecE on the phylogenetic tree of the RecT/Redβ superfamily, in- dicates that the RecE-RecT combination was probably the ancestral state for at least the RecT and EHAP1 families (Fig. 2). This implies that, on at least two occasions, the gene for λ endonuclease displaced the functionally analo- gous recE gene and became the adjacent gene to RecT (Fig. 2). That this displacement might have occurred by in situ insertion of a non-orthologous gene is suggested by the detection, on three separate occasions, of unusual rem- nants of pre-existing genes. The RecT/Redβ superfamily members, namely EHAP1 from the enterobacteria and PF161 from Borrelia hermsii, contain a small, C-terminal fragment of the core conserved domain of the ERF super- family, which is located C-terminal of their bona fide RecT/ Redβ domains. These fragments of the ERF protein are closely related to other ERF domains from related organ- isms and are unlikely to fold into the native conformation characteristic of the full-length ERF domain. For example the ERF fragment fused to the EHAP1 RecT/Redβ domain is closely related to the P22 phage ERF domain. This sug- gests that in each of these cases a RecT superfamily gene was inserted in frame into a pre-existing ERF gene leaving behind only a non-functional fragment of it (Fig. 2). In a very similar case, the bacterial RAD52-like protein from a Shiga toxin encoding temperate phage is fused to an ex- treme C-terminal fragment that is nearly identical to the C-terminal most portion of the P22 ERF protein. In this case, it appears that the pre-existing ERF gene was dis- placed through the insertion of a bacterial RAD52-like gene. Interestingly, and in the same vein, the RecT pro- teins from Bacillus species contain a short C-terminal acid- ic module that is missing in other RecT proteins, but is highly similar to the C-terminal region of SSBs, particular- ly those from Gram-positive bacteria (data not shown). This suggests that, at some stage in their evolution, the Ba- cillus recT gene protein has recombined with the gene cod- ing for SSB, which might even have resulted in a functional replacement of an SSB with an SSAP. Thus it appears likely that functionally equivalent genes may displace their analogs in operons via insertion into the same position. Conclusions We show that functionally similar SSAPs belong to at least three evolutionarily distinct superfamilies. We unify the Redβ and RecT proteins and their homologs, which have not been reported as being related at the sequence level, into a single superfamily, supporting the notion that these proteins share a similar mechanism of action. The second superfamily typified by the ERF proteins is predominantly found in bacteriophages and is also present on all circular plasmids from Borrelia, suggesting a role in the recombi- nation of these plasmids. The third superfamily, typified by the yeast RAD52 protein and previously detected only in eukaryotes, was shown to include bacterial and phage homologs and to contain a modified HhH domain. By comparing the gene neighborhoods of the SSAPs, we show that the predicted operons that include the SSAP genes evolve according to the "LEGO" principle. In these operons, the SSAP genes are linked to the genes for vari- ous DNAses and DNA repair related proteins, such as SSB Page 9 of 11 (page number not for citation purposes)