One Year Genome Evolution of Lausannevirus in Allopatric versus Sympatric Conditions

Abstract

Amoeba-resisting microorganisms raised a great interest during the last decade. Among them, some large DNA viruses present huge genomes up to 2.5 Mb long, exceeding the size of small bacterial genomes. The rate of genome evolution in terms of mutation, deletion, and gene acquisition in these genomes is yet unknown. Given the suspected high plasticity of viral genomes, the microevolution of the 346 kb genome of Lausannevirus, a member of Megavirales, was studied. Hence, Lausannevirus was co-cultured within the amoeba Acanthamoeba castellanii over one year. Despite a low number of mutations, the virus showed a genome reduction of 3.7% after 12 months. Lausannevirus genome evolution in sympatric conditions was investigated by its co-culture with Estrella lausannensis, an obligate intracellular bacterium, in the amoeba A. castellanii during one year. Cultures were split every 3 months. Genome sequencing revealed that in these conditions both, Lausannevirus and E. lausannensis, show stable genome, presenting no major rearrangement. In fact, after one year they acquired from 2 to 7 and from 4 to 10 mutations per culture for Lausannevirus and E. lausannensis, respectively. Interestingly, different mutations in the endonuclease encoding genes of Lausannevirus were observed in different subcultures, highlighting the importance of this gene product in the replication of Lausannevirus. Conversely, mutations in E. lausannensis were mainly located in a gene encoding for a phosphoenolpyruvate-protein phosphotransferase (PtsI), implicated in sugar metabolism. Moreover, in our conditions and with our analyses we detected no horizontal gene transfer during one year of co-culture.

Keywords: Chlamydiales; Lausannevirus; Marseilleviridae; Megavirales; experimental evolution; single nucleotide polymorphisms (SNPs).

Fig. 1.

—Lausannevirus evolution in allopatric conditions: DNA copies number among duplicates. The number of viral DNA copies was assessed by qPCR for both duplicates, La01 and La02. Both subcultures show similar profiles, with a constantly high number of viral DNA copies/l along the year of co-culture.

Fig. 2.

—Lausannevirus and E. lausannensis subcultures during one year of co-culture in A. castellanii: schema and DNA copies number among the different subcultures. (A) Schema of Lausannevirus and E. lausannensis co-culture. Lausannevirus and E. lausannensis were maintained in co-culture with A. castellanii in 75 cm² flasks. Each three months (blue spots) the co-cultures were split into two different subcultures. The virus and the bacteria DNA were extracted and stored. (B) Number of Lausannevirus DNA copies in co-cultures. The number of viral DNA copies assessed by qPCR each 3 months before co-culture split is represented here in a log scale for all eight subculture (A–H) along time. Subcultures present very different number of viral DNA copies/l along the year of co-culture. Two main profile, high and low, are distinct, at 12 months only subcultures C12 show an intermediate profile, subcultures D12, E12, F12, and H12 have high profile while subcultures A12, B12, and G12 show low profiles. (C) Number of E. lausannensis DNA copies in co-cultures. As for the virus, the number of bacterial DNA copies was assessed by qPCR each 3 months. All the eight subcultures show similar profile along the year of co-culture, with low number of DNA copies/l until 3 months after which all subcultures increase dramatically the number of DNA copies/l and stay high until the end of the year. (D) Schema of Lausannevirus co-culture in massive presence of E. lausannensis. Lausannevirus was kept in co-culture one year (144 passages) with the addition of E. Lausannensis to the co-culture after each passage. Each month the cultures were purified and stored.

Fig. 3.

—Circular representation of Lausannevirus mutations. The external circle represents Lausannevirus reference genome. Green circles from the outside to the inside: culture A12, culture C12, culture D12, culture E12, culture F12, culture H12. In blue and sky blue are genes in which a mutation (red) was detected. Zoom on the 195–200 kb region: mutations on two putative restriction endonucleases: mutations in positions 195949 (blue gene) and 195970 (sky blue gene) in subcultures C12, D12, and F12, mutation in position 195948 in subculture H12.

Fig. 4.

—Mutations distribution along giant viruses. Representation of orthologue groups among giant viruses. Red colors are the protein in which at least one mutation was observed in Lausannevirus allopatric or sympatric co-cultures. Most mutations occurred in regions relatively conserved among Marseilleviridae, but not conserved among all giant viruses.

Fig. 5.

—Translation of Lausannevirus variants putative restriction endonucleases and conserved domains detection. Homologous parts are highlighted by the same dotted blue part. The top translated variant, with mutations 195949/195970 shows no frameshift and carry the same conserved domain as the Lausannevirus reference genome. The deletion in position 195948 shows a correction of frameshift and encode for a longer protein compared with the Lausannevirus reference, with a larger region of homology with Insectomime putative restriction endonuclease (ISTM_159). In this variant, as in Insectomime, 4 DUF4379 domains and a restriction endonuclease domain were detected.

Fig. 6.

—Circular representation of E. lausannensis mutations. Mutations among the eight E. lausannensis subcultures are represented. The external circle represents E. lausannensis reference genome. Green circles from the outside to the inside: culture A12, culture B12, culture C12, culture D12, culture E12, culture F12, culture G12, and culture H12. Mutations are distributed along the whole genome and several subcultures present mutations in the same gene.

Fig. 7.

—Pyruvate generated between 0 and 60 min by the PtsI WT and the PtsI variant 352605. Amount (nmol) of pyruvate generated between 0 and 60 min. Among the WT (orange) and the positive control (blue) no significant difference were detected (P-value = 0.37). PtsI variant 352605 (green) showed significant (*) difference in pyruvate generation when compared with the WT (P-value = 0.01).