Complete Genome Sequence of Francisella philomiragia ATCC 25017

Abstract

Francisella philomiragia is a saprophytic gammaproteobacterium found only occasionally in immunocompromised individuals and is the nearest neighbor to the causative agent of tularemia and category A select agent Francisella tularensis. To shed insight into the key genetic differences and the evolution of these two distinct lineages, we sequenced the first complete genome of F. philomiragia strain ATCC 25017, which was isolated as a free-living microorganism from water in Bear River Refuge, Utah.

GENOME ANNOUNCEMENT

The genome of Francisella philomiragia subsp. philomiragia ATCC 25017 was sequenced using traditional Sanger sequencing of 3-kb shotgun libraries. The initial draft assembly was comprised of 40,596 reads resulting in 116 contigs (∼18-fold coverage), a rather large number given the small (∼2-Mb) anticipated genome size, due in part to a large number of repetitive elements. Genome closure and polishing were accomplished using an additional 469 targeted sequencing reads to join contigs, close gaps, and resolve all repetitive elements. The resulting finished-quality (2) completed genome of F. philomiragia is comprised of a chromosome of 2,045,775 bp in length (32.0% G+C content) along with a small, 3,936-bp, cryptic plasmid, pFPHIo1 (28.4% G+C content), which encodes only four putative hypothetical proteins with unknown function. While the G+C contents are similar among all 10 currently sequenced Francisella tularensis genomes, F. philomiragia has a larger genome (2.045 Mb versus 1.9 Mb) than do its F. tularensis near neighbors.

The F. philomiragia chromosome includes 1,966 genes and 7 pseudogenes, which are comparable to the numbers of coding sequences (1,898) and pseudogenes (14) in the opportunistic pathogenic F. tularensis subsp. novicida genome (3), whereas the chromosome of pathogenic F. tularensis subsp. tularensis strain SCHU carries a much higher load of pseudogenes (200) with concomitantly fewer (1,852) regular genes (6). This observation is consistent with the process of ongoing genome reduction seen in recently evolved pathogens adapting to a specialized lifestyle (1, 7).

The F. philomiragia chromosome carries a large number of insertion sequence (IS) elements, including several previously described IS families: IS30 (2 copies), ISFtu1 (2 copies), ISFtu2 (34 copies), ISFtu3 (3 copies), ISFtu6 (1 copy), IS1106 (2 copies), IS1016 (13 copies), IS4 (17 copies), IS1301 (3 copies), and IS200 (1 copy). ISFtu1, ISFtu2, SFtu3, and ISFtu6 are also shared with the other sequenced F. tularensis strains (3), although the copy number varies among strains. Notably, ISFtu1 has been used as a marker to discriminate between highly pathogenic and less pathogenic F. tularensis subtypes (5).

The genome of F. philomiragia also appears to harbor substantial differences with respect to putative virulence factors described in F. tularensis Schu S4. For example, the intracellular growth locus proteins A, B, C, and D (Fphi_1364, Fphi_1365, Fphi_1366, and Fphi_1367, respectively), encoded within a large 33.9-kb duplicated pathogenicity island first described in pathogenic F. tularensis (8), are also present in a homologous genomic segment which is found only once in the genome of Francisella philomiragia. Another F. tularensis virulence factor, phospholipase C, has been shown to regulate cell membrane biogenesis and is involved in interactions with the eukaryotic host (4). Interestingly, in F. philomiragia, this protein (Fphi_0739) shares over 95% identity with other Francisella species and 50% identity with phospholipase C in Ktedonobacter racemifer and in Bacillus, Streptomyces, Burkholderia, and Corynebacterium species. A more in-depth comparative analysis of the genome and metabolic pathways found in F. philomiragia and F. tularensis may lead us to identify additional factors responsible for the high pathogenicity found only in some F. tularensis subspecies.

Nucleotide sequence accession number.

The nucleotide sequence of the finished genome of F. philomiragia subsp. philomiragia was deposited in GenBank under accession number NC_010336.1.