Francisella tularensis is one of the most infectious human pathogens known. In the past, both the former Soviet Union and the US had programs to develop weapons containing the bacterium. We report the complete genome sequence of a highly virulent isolate of F. tularensis (1,892,819 bp). The sequence uncovers previously uncharacterized genes encoding type IV pili, a surface polysaccharide and iron-acquisition systems. Several virulence-associated genes were located in a putative pathogenicity island, which was duplicated in the genome. More than 10% of the putative coding sequences contained insertion-deletion or substitution mutations and seemed to be deteriorating. The genome is rich in IS elements, including IS630 Tc-1 mariner family transposons, which are not expected in a prokaryote. We used a computational method for predicting metabolic pathways and found an unexpectedly high proportion of disrupted pathways, explaining the fastidious nutritional requirements of the bacterium. The loss of biosynthetic pathways indicates that F. tularensis is an obligate host-dependent bacterium in its natural life cycle. Our results have implications for our understanding of how highly virulent human pathogens evolve and will expedite strategies to combat them.

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (

Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. mediasiatica, F. tularensis subsp. holarctica, and F. tularensis subsp. novicida. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensis subsp. tularensis Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis, and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e., F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of F. tularensis subsp. tularensis and F. tularensis subsp. mediasiatica was apparent. Strains from Japan clustered separately, as did strains of F. tularensis subsp. novicida. Eight regions of difference (RD) 0.6 to 11.5 kb in size, altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies F. tularensis subsp. holarctica and the highly virulent subspecies F. tularensis subsp. tularensis. One of these regions, RD1, allowed for the first time the development of an F. tularensis-specific PCR assay that discriminates each of the four subspecies.

Colorectal cancer (CRC) is the second most common cause of cancer death in the western world. An effective screening program leading to early detection of disease would severely reduce the mortality of CRC. Alterations in the gut microbiota have been linked to CRC, but the potential of microbial markers for use in CRC screening has been largely unstudied. We used a nested case–control study of 238 study subjects to explore the use of microbial markers for clbA+ bacteria harboring the pks pathogenicity island, afa‐C+ diffusely adherent Escherichia coli harboring the afa‐1 operon, and Fusobacterium nucleatum in stool as potential screening markers for CRC. We found that individual markers for clbA+ bacteria and F. nucleatum were more abundant in stool of patients with CRC, and could predict cancer with a relatively high specificity (81.5% and 76.9%, respectively) and with a sensitivity of 56.4% and 69.2%, respectively. In a combined test of clbA+ bacteria and F. nucleatum, CRC was detected with a specificity of 63.1% and a sensitivity of 84.6%. Our findings support a potential value of microbial factors in stool as putative noninvasive biomarkers for CRC detection. We propose that microbial markers may represent an important future screening strategy for CRC, selecting patients with a “high‐risk” microbial pattern to other further diagnostic procedures such as colonoscopy.

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.