Evolutionary genomics of a temperate bacteriophage in an obligate intracellular bacteria (Wolbachia)

Abstract

Genome evolution of bacteria is usually influenced by ecology, such that bacteria with a free-living stage have large genomes and high rates of horizontal gene transfer, while obligate intracellular bacteria have small genomes with typically low amounts of gene exchange. However, recent studies indicate that obligate intracellular species that host-switch frequently harbor agents of horizontal transfer such as mobile elements. For example, the temperate double-stranded DNA bacteriophage WO in Wolbachia persistently transfers between bacterial coinfections in the same host. Here we show that despite the phage's rampant mobility between coinfections, the prophage's genome displays features of constraint related to its intracellular niche. First, there is always at least one intact prophage WO and usually several degenerate, independently-acquired WO prophages in each Wolbachia genome. Second, while the prophage genomes are modular in composition with genes of similar function grouping together, the modules are generally not interchangeable with other unrelated phages and thus do not evolve by the Modular Theory. Third, there is an unusual core genome that strictly consists of head and baseplate genes; other gene modules are frequently deleted. Fourth, the prophage recombinases are diverse and there is no conserved integration sequence. Finally, the molecular evolutionary forces acting on prophage WO are point mutation, intragenic recombination, deletion, and purifying selection. Taken together, these analyses indicate that while lateral transfer of phage WO is pervasive between Wolbachia with occasional new gene uptake, constraints of the intracellular niche obstruct extensive mixture between WO and the global phage population. Although the Modular Theory has long been considered the paradigm of temperate bacteriophage evolution in free-living bacteria, it appears irrelevant in phages of obligate intracellular bacteria.

Figure 1. Prophage WO genomes are modular.

A schematic of gene synteny across the prophage WO genomes is depicted. Complete WO prophage sequences are available with the exception of wPip2 and wPip3, as the wPip genome sequence was artificially connected between genes within these two prophages . These two prophages are treated as separate and complete. The two prophages from wCauB have been shown to be excisable by the mapping of their att sites in conjunction with visualizing phage particles , . Inverse PCR and sequencing analysis showed that WOCauB2 is conjoined between the integrase B2gp1 and the ankyrin repeat protein B2gp47, and WOCauB3 is conjoined between the integrase B3gp1 and the putative SpvB family toxin B3gp45 and hypothetical protein encoding B3gp46 genes . WO haplotypes from wMel , wPip , and wVitA are presumed excisable due to observations of lytic phage particles in each system. Genes are colored based on functional type and homology. Bright pink: integrase/recombinase; Red: Ankyrin-repeat protein; Turquoise: Replication module; Purple: Head module; Blue: Baseplate module; Orange: Putative virulence factors; Green: Tail module; Yellow: Transposases; Light pink: Holliday junction resolvasome/endonuclease; Grey: DNA methylase, Light teal: SNF2 helicase, Dark teal: lysozyme. The numbers above genes refer to the locus tag of that gene in the published genome.

Figure 2. The core genome of prophage WO consists only of head and baseplate genes.

The percentage of prophage WO genomes (N = 16) containing genes present in the active phage genome, WOCauB2, is depicted. Percentages were calculated for both all WO genomes (black) and only WO genomes with tail genes (grey). A gene map of WOCauB2 is shown above the plot with colors corresponding to the labels in Figure 1.

Figure 3. Unique genes in WO genomes are rare and scattered across functional modules.

(A) The percentage of genes specific to a single prophage WO haplotype was calculated. (B) The percentage of unique genes present in each functional region across all WO prophages and standard error was identified by calculating the number of unique genes divided by the number of genes in the module.

Figure 4. WO flanking regions contain conserved gene sets.

The prophages WOVitA2, WOMelB, and WORiA are flanked on one end by a segment of genes that is conserved on a Rickettsial plasmid (blue). In WOMelB and WORiA, a second conserved gene set (green), comprised of transcriptional regulators and the DNA repair gene radC, is found downstream of the Rickettsia gene homologs. This region is the prophage-flanking region in WOPip1 and WOVitA1. A third conserved gene segment (red) of ankyrin repeat proteins, a heat shock protein, and a conserved hypothetical protein, is found in WOVitA1 and flanking the prophage terminal gene in WORiB. Light gray: RepA; Dark gray: SNF2-family helicase; Black: patatin.

Figure 5. Mean genetic distance of WO and Wolbachia genes.

The average genetic distance and standard error was calculated for genes across Wolbachia prophage WO haplotypes. The values for the hypervariable Wolbachia surface protein gene wsp and the highly conserved Wolbachia housekeeping genes coxA and ftsZ are provided for comparison. Black bars represent genes having a predicted function while gray bars represent genes for which no function can be predicted.