The repABC operon is not only important because it is the replication-partition system of repABC plasmids, a common component of Alphaproteobacteria species, but because it is also the replication-partition system of some secondary chromosomes in Alphaproteobacteria species. Our present analyses functioned at two levels: within the repABC operon and between repABC operons in those cases where several repABC operons coexisted in the same genome. We did not find a single history within the repABC operon; clearly, each protein had its own phylogeny. This is somewhat surprising, since repA, repB, and repC form an operon, and it would seem that they should have similar histories if the entire operon had been horizontally transferred. Instead, even RepA and RepB, which compose the partition system and physically interact, had different phylogenies. This contrast with a recent work in which relaxase sequences were used as tools for classification of conjugative systems. In that study it was found that relaxases and the IV coupling proteins (T4CP), which map next to each other and belong to a minimal gene set that allows plasmid to be conjugally transmitted, evolve congruently for long periods of time . Thus, it seems that compared with some elements of the transfer machinery the repABC replication-partition system is highly diverse.
Quite notably, every single gene of this operon presented evidence of horizontal gene transfer. In situ gene displacement is a likely process behind this, since the structure of the repABC operon is completely conserved. We think in situ gene displacement could have occurred through homologous recombination, as we found homologous recombination events across the 3 rep genes. Although in situ gene displacement appears unlikely, there is evidence that shows that this process is not that scarce. Omelchenko et al found that within the bacterial operons they had analyzed in situ gene displacement was a frequent event . A striking difference between in situ gene displacement and other types of horizontal gene transfer events is that the former leaves intact the operon structure, so that, the operon is completely functional.
The proteins differed not only at the topological level, but also at the level of functional restriction. RepA and RepC, which belong to different systems, were under similar levels of functional restriction, suggesting that key elements of the partitioning and replication systems are under similar functional restrictions. In contrast, RepB had a very different level of functional restriction. We also found different levels of functional restrictions within proteins. For example, the ATPase domain of RepA (Figure 3, family MipZ), which forms a complex with the chromosome partitioning protein and is indispensable for partitioning, presented the lowest substitution rates. As well the only recombination event presented in repA did not affect the ATPase domain but a relatively unconserved part of the gene. Therefore, it seems that the different proteins, and even the different parts of the proteins themselves, are under different functional and/or structural constraints. Of the three genes studied, repA was the most conserved and might have the highest expression level. This is not unexpected, as RepA is known to have several functions, and its expression is required in both the presence and absence of partition, suggesting the need for high-level translation in order to maintain sufficient RepA levels. In contrast, repC, which is a replication initiator protein, had the lowest CAI values, perhaps due to the higher levels of homologous recombination in this gene (see below). Horizontal gene transfer could be very important in allowing the variability of this operon. Indeed, if horizontal gene transfer had not affected the genes within the operon, these genes would have to have a single evolutionary history. Instead, we found that the reverse was true. The proteins encoded in those genes not only presented different phylogenies, they also had different functional restrictions, even within the proteins themselves, and the CAI values differed among the genes. Given the presence of differences at several levels, it is very logical to think that horizontal gene transfer has unconnected the various portions of the operon, allowing each part to have a particular evolutionary history. In this way, genes with very different functional restrictions could be located next to each other, as seen for repB and repA.
The existence of multiple repABC operons located on different replicons in the same genome implies the presence of different incompatibility groups. We herein showed that when multiple repABC operons coexisted in the same genome, they were well differentiated from one another. We did not find evidence of homologous recombination in these cases; this is not unexpected, since homologous recombination would homogenize the sequences, meaning that the different groups would no longer be compatible with each other. The intergenic region, which encodes a small antisense RNA (a very important determinant for incompatibility), was highly conserved and found to be under high functional restriction, yet it did not have any invariant sites. Although this sequence has changed only minimally due to functional restrictions, it has still accumulated sufficient changes to allow the coexistence of the different incompatibility groups. In agreement with our within-operon analysis, repA and repC were highly conserved, with repC being the most highly conserved between operons (it had the smallest average distance). As mentioned above, repC also had the most homologous recombination events. This suggests that homologous recombination might be reducing the divergence of repC, potentially also explaining the low CAI values for this gene (homologous recombination would be erasing any improvement in the CAI values). In a report on the genome sequence of R. leguminosarum, Young and coworkers suggested that a recent recombination event had taken place, and divergence of RepC was not critical for plasmid compatibility . Here, one of the recombination events detected in repC involved the sequence from pRL8, which is a plasmid of R. leguminosarum 3841.
Different repABC operons had distinct levels of adaptation to their host genome, with no two repABC operons presenting the same CAI values. We think that amelioration might be playing a role in the adaptation of repABC operons to their hosts. Plasmids p42a and p42d were suggested to be newly acquired plasmids based on their lower GC values, poor conservation, and poor functional connectivity with the rest of the genome . These two plasmids had the worst CAI values, implying that they are not well adapted to their host's genome. In contrast, the operon from p42f, which appeared to be the oldest plasmid harbored within R. etli CFN42, had the highest CAI values, suggesting that this operon is highly adapted. These findings indicate that the longer a repABC operon coexists with its host genome, the more adapted the operon becomes. This may result in more effective replication and partitioning processes. As well plasmids, which had the most adapted operons, presented essential genes as well; for instance plasmids pRL11, pRL12, and pRL10, which all have essential genes , had the operons with higher CAI values than the rest of plasmid of R. leguminosarum 3841.