Unexpectedly high mutation rate of a deep-sea hyperthermophilic anaerobic archaeon

Abstract

Deep-sea hydrothermal vents resemble the early Earth, and thus the dominant Thermococcaceae inhabitants, which occupy an evolutionarily basal position of the archaeal tree and take an obligate anaerobic hyperthermophilic free-living lifestyle, are likely excellent models to study the evolution of early life. Here, we determined that unbiased mutation rate of a representative species, Thermococcus eurythermalis, exceeded that of all known free-living prokaryotes by 1-2 orders of magnitude, and thus rejected the long-standing hypothesis that low mutation rates were selectively favored in hyperthermophiles. We further sequenced multiple and diverse isolates of this species and calculated that T. eurythermalis has a lower effective population size than other free-living prokaryotes by 1-2 orders of magnitude. These data collectively indicate that the high mutation rate of this species is not selectively favored but instead driven by random genetic drift. The availability of these unusual data also helps explore mechanisms underlying microbial genome size evolution. We showed that genome size is negatively correlated with mutation rate and positively correlated with effective population size across 30 bacterial and archaeal lineages, suggesting that increased mutation rate and random genetic drift are likely two important mechanisms driving microbial genome reduction. Future determinations of the unbiased mutation rate of more representative lineages with highly reduced genomes such as Prochlorococcus and Pelagibacterales that dominate marine microbial communities are essential to test these hypotheses.

Fig. 1. Experimental determination of the unbiased mutation rate of the Thermococcus eurythermalis A501 is challenging because this archaeon has unusual physiology (i.e., obligate anaerobic and obligate hyperthermophilic).

A The preparation of anaerobic high temperature tolerant gelrite plate. After sterilization and polysulfide addition via syringe, the plates are made in an anaerobic chamber. B The incubation of the strain T. eurythermalis A501 at 85 °C in liquid medium. C The initiation of mutation accumulation (MA) by spreading cells from a single founding colony to 100 lines. Plates are placed in an anaerobic jar for incubation in strictly anaerobic condition at 85 °C. D The MA process followed by whole-genome sequencing and data analysis. Single colony of each line is transferred to a new plate for N times (here N = 20). E Base-substitution mutations and insertion/deletion mutations across the whole genome of T. eurythermalis. The dashed vertical line separates the chromosome and plasmid. The height of each bar represents the number of base-substitution mutations across all MA lines within 10 kbp window. Green and red triangles denote insertion and deletion, respectively. The locus tags of the 14 genes with statistical enrichment of mutations are shown.

Fig. 2. The scaling relationship involving the base-substitution mutation rate per cell division per site (µ), the estimated effective population size (N_e), and genome size across 28 bacterial and two archaeal species.

All three traits’ values were logarithmically transformed. The mutation rates of these species are all determined with the mutation accumulation experiment followed by whole-genome sequencing of the mutant lines. The mutation rate of species numbered 1–29 (blue) is collected from literature and that of the species 30 (red) is determined in the present study. Among the numbered species shown in the figure, the species #6 Haloferax volcanii is facultative anaerobic halophilic archaeon, and the species #30 is an obligate anaerobic hyperthermophilic archaeon. A The scaling relationship between µ and N_e. B The scaling relationship between µ and genome size. C The scaling relationship between genome size and N_e. Numbered data points 21–29 are not shown in A and C because of the lack of population dataset for estimation of N_e. The dashed gray lines and blue lines represent the generalized linear model (GLM) regression and the phylogenetic generalized least square (PGLS) regression, respectively. The Bonferroni adjusted outlier test for the GLM regression show that #7 Janthinobacterium lividum is an outlier in the scaling relationship between µ and N_e, and #9 Mesoplasma florum is an outlier in the scaling relationship between genome size and N_e. No outlier was identified in the PGLS regression results.