Molecular Evolutionary Consequences of Niche Restriction in Francisella tularensis, a Facultative Intracellular Pathogen

Abstract

Francisella tularensis is a potent mammalian pathogen well adapted to intracellular habitats, whereas F. novicida and F. philomiragia are less virulent in mammals and appear to have less specialized lifecycles. We explored adaptations within the genus that may be linked to increased host association, as follows. First, we determined the genome sequence of F. tularensis subsp. mediasiatica, the only subspecies that had not been previously sequenced. This genome, and those of 12 other F. tularensis isolates, were then compared to the genomes of F. novicida (three isolates) and F. philomiragia (one isolate). Signs of homologous recombination were found in ∼19.2% of F. novicida and F. philomiragia genes, but none among F. tularensis genomes. In addition, random insertions of insertion sequence elements appear to have provided raw materials for secondary adaptive mutations in F. tularensis, e.g. for duplication of the Francisella Pathogenicity Island and multiplication of a putative glycosyl transferase gene. Further, the five major genetic branches of F. tularensis seem to have converged along independent routes towards a common gene set via independent losses of gene functions. Our observations suggest that despite an average nucleotide identity of >97%, F. tularensis and F. novicida have evolved as two distinct population lineages, the former characterized by clonal structure with weak purifying selection, the latter by more frequent recombination and strong purifying selection. F. tularensis and F. novicida could be considered the same bacterial species, given their high similarity, but based on the evolutionary analyses described in this work we propose retaining separate species names. The intracellular bacterium Francisella tularensis causes the disease tularemia in various mammals, including humans, and is highly infectious (so infectious that highly virulent forms of the pathogen were developed as biological aerosol weapons during the Cold War). Little is known about where F. tularensis resides in nature and how it evolved but, intriguingly, closely related Francisella bacteria are less dangerous. Therefore, we have explored the evolutionary events that shaped F. tularensis by analyzing 17 Francisella genome sequences. Its evolution appears to have involved many losses of metabolic functions and random mutations, with little exchange of genetic material among F. tularensis strains. Furthermore, increased host association appears to have irreversibly separated F. tularensis populations from other populations of Francisella bacteria. This study provides new information on the processes whereby relatively harmless Francisella bacteria evolved into aggressive invaders of mammalian cells. Our findings support previous proposals that identification of distinct population lineages provides meaningful species boundaries among bacteria.