The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism

Abstract

Genome mosaicism in temperate bacterial viruses (bacteriophages) is so great that it obscures their phylogeny at the genome level. However, the precise molecular processes underlying this mosaicism are unknown. Illegitimate recombination has been proposed, but homeologous recombination could also be at play. To test this, we have measured the efficiency of homeologous recombination between diverged oxa gene pairs inserted into lambda. High yields of recombinants between 22% diverged genes have been obtained when the virus Red Gam pathway was active, and 100 fold less when the host Escherichia coli RecABCD pathway was active. The recombination editing proteins, MutS and UvrD, showed only marginal effects on lambda recombination. Thus, escape from host editing contributes to the high proficiency of virus recombination. Moreover, our bioinformatics study suggests that homeologous recombination between similar lambdoid viruses has created part of their mosaicism. We therefore propose that the remarkable propensity of the lambda-encoded Red and Gam proteins to recombine diverged DNA is effectively contributing to mosaicism, and more generally, that a correlation may exist between virus genome mosaicism and the presence of Red/Gam-like systems.

Figure 1. Recombination between the two homeologous oxa sequences flanking the λ pL promoter leads to a phenotypic switch.

In λ red- gam-, transcription from the pL promoter proceeds rightward and the red and gam genes are not transcribed. Recombination between the two homeologous sequences leads to inversion of the pL-N segment causing a selectable phenotype (see Text for details). Not drawn to scale.

Figure 2. Frequency of recombinants in the RecABCD and the Red pathways, at different levels of sequence divergence, determined by single step growth of phages.

Every bar shows the mean and standard deviation deduced from at least three experiments.

Figure 3. Generation of an inversion between the oxa sequences.

Left panel: two successive, non reciprocal events: Oxa sequences are shown with oriented boxes, dark and light gray. Step 1, a broken piece containing the rightward copy of oxa recombines with intact DNA, and generates an unviable molecule (step2), which in turn recombines its leftward oxa copy with another oxa sequence (step 3). This other broken piece eventually, but not necessarily, consists of the rest of the broken piece shown in step 1. Recombination gives rise to a viable product where the intermediate sequences have been inverted, and the two oxa genes are hybrids (step 4). A and R designate the leftmost and righmost genes of λ. Right panel: one reciprocal event. A crossing over is initiated intra-molecularly between the oxa sequences (step 1), and gives rise to the inverted configuration (step 2).

Figure 4. Sequence analysis of recombinant products between 22% diverged oxa.

A and C: size distribution of the strictly conserved segments (blocks) of the oxa7/oxa5 alignment (see figure S1, panel B, for the alignment), and number of joints in each size category for recombinants formed in the RecABCD (A) or the Red (B) pathway. B and D: Positional organisation of the joints formed in the RecABCD (B) or the Red (D) pathway along the oxa ORF alignement, 1 is the start position. The upper curve (and the rightward y axis) describes the local percentage of identity along the two sequences, using a sliding window of 40 bp.

Figure 5. Evolutionary history of event between diverged viruses A and B, produces virus C which is similar to A but for a patch of DNA (hit) coming from B.

Alignment of phage B to C reveals at the border of the patch two regions of intermediate similarity (shoulders, here drawn in grey). Below is shown the % identity profile of the B to C virus alignment.

Figure 6. Both λ Nec3 and λ Nec6 produce rolling circle intermediates.

DNA extracted during a kinetics of λ infection was loaded on a gel (30 µl), besides a λ ladder size marker (shown as L, 1 ng in the middle lane, 10 ng on the right side lane). UV photograph of the gel is shown in the left panel, and Southern blot in the right panel (the last lane was omitted for blotting). Mo, Di and RC: monomer, dimer and rolling-circle forms of λ respectively. C: E. coli C600 chromosomal DNA.