Comparative and evolutionary analysis of the bacterial homologous recombination systems

Abstract

Homologous recombination is a housekeeping process involved in the maintenance of chromosome integrity and generation of genetic variability. Although detailed biochemical studies have described the mechanism of action of its components in model organisms, there is no recent extensive assessment of this knowledge, using comparative genomics and taking advantage of available experimental data on recombination. Using comparative genomics, we assessed the diversity of recombination processes among bacteria, and simulations suggest that we missed very few homologs. The work included the identification of orthologs and the analysis of their evolutionary history and genomic context. Some genes, for proteins such as RecA, the resolvases, and RecR, were found to be nearly ubiquitous, suggesting that the large majority of bacterial genomes are capable of homologous recombination. Yet many genomes show incomplete sets of presynaptic systems, with RecFOR being more frequent than RecBCD/AddAB. There is a significant pattern of co-occurrence between these systems and antirecombinant proteins such as the ones of mismatch repair and SbcB, but no significant association with nonhomologous end joining, which seems rare in bacteria. Surprisingly, a large number of genomes in which homologous recombination has been reported lack many of the enzymes involved in the presynaptic systems. The lack of obvious correlation between the presence of characterized presynaptic genes and experimental data on the frequency of recombination suggests the existence of still-unknown presynaptic mechanisms in bacteria. It also indicates that, at the moment, the assessment of the intrinsic stability or recombination isolation of bacteria in most cases cannot be inferred from the identification of known recombination proteins in the genomes.

Figure 1. Models for the Mode of Action of the Main Homologous Recombination Proteins in E. coli at ssDNA Gaps or dsDNA Ends

Enzymes of known biochemical activities are shown. The presynaptic steps result in the formation of a RecA filament. At gaps, this step requires RecJ, RecF, RecO, and RecR: the 5′ ssDNA exonuclease RecJ enlarges the ssDNA region (possibly with the help of various helicases, as no specific helicase is required for gap repair); RecF, RecO, and RecR promote RecA binding to SSB-coated DNA. At dsDNA ends, RecBCD (AddAB in B. subtilis) degrades DNA until it encounters a χ site; its helicase-nuclease activity is then modified to produce a 3′-ended ssDNA, to which it loads RecA. The synaptic step (homology search and strand exchange) is always performed by RecA and results in the formation of a Holliday junction (X structure). The postsynaptic steps are the migration and the resolution of Holliday junctions. Migration can be performed by RuvAB or by RecG, and resolution is made by RuvC (RecU in B. subtlis; RuvC forms a complex with RuvAB in E. coli). In addition, RecBCD-mediated recombination is always coupled with PriA-dependent replication restart. Antirecombinases are not shown: UvrD and MutLS prevent by different means the strand exchange reaction. In recBC mutants, the presynaptic steps of dsDNA end repair can be catalyzed by the helicase RecQ and the gap repair proteins RecJ and RecFOR, a reaction that is prevented by SbcB (and SbcCD) nucleases.

Figure 2. Probable Presence, Putative, and Unlikely Presence of Recombination-Associated Genes in the Studied Genomes

Black indicates presence is probably, grey indicates putative presence, and white indicates presence is unlikely. F indicates that the gene is present in the genome but contains frame shifts (genes with known programmed frame shifts, introns, and inteins are indicated, as regular genes, in black).

Figure 3. Distribution of Some Recombination Genes in a Phylogenetic Tree of Bacteria

Tree adapted from [99]. The position of Pirellula and Fusobacterium are still unclear [100].

Figure 4. Unrooted Phylogenetic Tree of the RecD Protein

The dotted line separates genomes containing RecBCD from the ones containing only RecD. The tree was constructed using Tree-Puzzle, using the JTT+Γ model with eight classes [74]. Bootstraps were made using SEQBOOT and CONSENSE from the PHYLIP package [101]. C. acetobutylicum, Clostridium acetobutylicum; C. tepidum, Chlorobium tepidum; C. violaceum, Chromobacterium violaceum; D. vulgaris, Desulfovibrio vulgaris; E. carotovora, Erwinia carotovora; E. faecalis, Enterococcus faecalis; L. plantarum, Lactobacillus plantarum; L. lactis, Lactococcus lactis; P. multocida, Pasteurella multocida; M. mobile, Mycoplasma mobile; M. florum, Mesoplasma florum; M. mycoides, Mycoplasma mycoides; M. pulmonis, Mycoplasma pulmonis; P. maritima, Procholorcoccus maritima; S. enterica, Salmonella enterica; S. oneidensis, Shewanella oneidensis; X. fastidiosa, Xylella fastidiosa.

Figure 5. Regression Lines of the Substitution Rates of the Recombination Proteins Plotted against the Substitution Rates of RecA

RecA is the slowest and most ubiquitous of these proteins and its substitution rates are the x-axis of the plot (dashed lines indicate the RecA identity line). The regression was forced to pass through zero at intercept, and the slopes of the lines indicate the relative rapidity of the protein's evolution relative to that of RecA (varies from 1.3 for RuvB to 4.4 to RecB); the points associated with mollicutes were removed because we found these to evolve proportionally faster in RecA than in the other proteins.

Figure 6. Results of the Simulations of Protein Evolution following the Phylogenetic Tree of RecA Using the JTT+Γ Model with Eight Classes and an α of 0.59

We used Seq-Gen to evolve protein sequences with regularly spaced scaling factors (100 experiments for each scaling factor) and analyzed for each experience which sequences showed less than 40% similarity.