The complete genome sequence of Francisella tularensis, the causative agent of tularemia

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.

[1]  M. Chandler,et al.  Insertion Sequences , 1998, Microbiology and Molecular Biology Reviews.

[2]  A. M. Hood.  Virulence factors of Francisella tularensis , 1977, Journal of Hygiene.

[3]  J. Mager,et al.  STUDIES ON THE NUTRITION OF PASTEURELLA TULARENSIS , 1955, Journal of bacteriology.

[4]  M. Horwitz,et al.  Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages , 2004, Infection and Immunity.

[5]  Peter D. Karp,et al.  Using functional and organizational information to improve genome-wide computational prediction of transcription units on pathway-genome databases , 2004, Bioinform..

[6]  R. Knights,et al.  Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells. , 1993, Science.

[7]  J. Mager.  The stabilizing effect of spermine and related polyamines and bacterial protoplasts. , 1959, Biochimica et biophysica acta.

[8]  Anton J. Enright,et al.  Protein families and TRIBES in genome sequence space. , 2003, Nucleic acids research.

[9]  A. Sjöstedt,et al.  Analysis of 16S ribosomal DNA sequences of Francisella strains and utilization for determination of the phylogeny of the genus and for identification of strains by PCR. , 1994, International journal of systematic bacteriology.

[10]  Peter D. Karp,et al.  A Bayesian method for identifying missing enzymes in predicted metabolic pathway databases , 2004, BMC Bioinformatics.

[11]  D. Thanassi,et al.  Presence of Pili on the Surface of Francisella tularensis , 2004, Infection and Immunity.

[12]  N. Cianciotto,et al.  The Legionella pneumophila iraAB Locus Is Required for Iron Assimilation, Intracellular Infection, and Virulence , 2000, Infection and Immunity.

[13]  R A Gibbs,et al.  A "double adaptor" method for improved shotgun library construction. , 1996, Analytical biochemistry.

[14]  Jeffrey R. Barker,et al.  MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[15]  R. E. Chamberlain,et al.  EVALUATION OF LIVE TULAREMIA VACCINE PREPARED IN A CHEMICALLY DEFINED MEDIUM. , 1965, Applied microbiology.

[16]  Na Zhang,et al.  A Francisella tularensis Pathogenicity Island Required for Intramacrophage Growth , 2004, Journal of bacteriology.

[17]  Peter D. Karp,et al.  MetaCyc: a multiorganism database of metabolic pathways and enzymes. , 2004, Nucleic acids research.

[18]  S. Andersson,et al.  A phylogenomic study of endosymbiotic bacteria. , 2004, Molecular biology and evolution.

[19]  B. Palsson,et al.  An expanded genome-scale model of Escherichia coli K-12 (iJR904 GSM/GPR) , 2003, Genome Biology.

[20]  S. Salzberg,et al.  Improved microbial gene identification with GLIMMER. , 1999, Nucleic acids research.

[21]  I. Golovliov,et al.  An Attenuated Strain of the Facultative Intracellular Bacterium Francisella tularensis Can Escape the Phagosome of Monocytic Cells , 2003, Infection and Immunity.

[22]  Sean R. Eddy,et al.  Profile hidden Markov models , 1998, Bioinform..

[23]  R. Titball,et al.  The efficacy of ciprofloxacin and doxycycline against experimental tularaemia. , 1998, The Journal of antimicrobial chemotherapy.

[24]  B. Barrell,et al.  Massive gene decay in the leprosy bacillus , 2001, Nature.

[25]  W. Braun,et al.  STUDIES ON THE VARIATION OF BACTERIUM TULARENSE , 1951, Journal of bacteriology.

[26]  Nature Genetics , 1991, Nature.

[27]  P. Bork,et al.  A new family of carbon‐nitrogen hydrolases , 1994, Protein science : a publication of the Protein Society.

[28]  M. Simmonds,et al.  Genome sequence of Yersinia pestis, the causative agent of plague , 2001, Nature.

[29]  I. Golovliov,et al.  A method for allelic replacement in Francisella tularensis. , 2003, FEMS microbiology letters.

[30]  R. B. Narayanan,et al.  Growth of Francisella tularensis LVS in macrophages: the acidic intracellular compartment provides essential iron required for growth , 1995, Infection and immunity.

[31]  A. Sjöstedt,et al.  Discrimination of Human Pathogenic Subspecies of Francisella tularensis by Using Restriction Fragment Length Polymorphism , 2003, Journal of Clinical Microbiology.

[32]  J. Mager,et al.  An Endogenously Produced Substance Essential for Growth Initiation of Pasteurella tularensis , 1967 .

[33]  E. Hickey,et al.  An iron- and fur-repressed Legionella pneumophila gene that promotes intracellular infection and encodes a protein with similarity to the Escherichia coli aerobactin synthetases , 1997, Infection and immunity.

[34]  T. Doak.  A proposed superfamily of transposase-related genes: new members in transposon-like elements of ciliated protozoa and a common "D35E" motif , 1994 .

[35]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.

[36]  Peter D. Karp,et al.  The Pathway Tools software , 2002, ISMB.

[37]  A. Fortier,et al.  Heat stress alters the virulence of a rifampin-resistant mutant of Francisella tularensis LVS , 1995, Infection and immunity.

[38]  M. Telepnev,et al.  Factors affecting the escape of Francisella tularensis from the phagolysosome. , 2004, Journal of medical microbiology.

[39]  C. Mody,et al.  A mutation in the mip gene results in an attenuation of Legionella pneumophila virulence. , 1990, The Journal of infectious diseases.

[40]  D E FLEMING,et al.  STUDIES ON THE PHYSIOLOGY OF VIRULENCE OF PASTEURELLA TULARENSIS I , 1955, Journal of bacteriology.

[41]  C. Fraser,et al.  Complete genome sequence of the Q-fever pathogen Coxiella burnetii , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[42]  Philip K. Russell,et al.  Tularemia as a biological weapon: medical and public health management. , 2001, JAMA.

[43]  J. Mager.  A Factor required for Growth Initiation of Pasteurella tularensis , 1964, Nature.

[44]  M. Telepnev,et al.  Francisella tularensis inhibits Toll‐like receptor‐mediated activation of intracellular signalling and secretion of TNF‐α and IL‐1 from murine macrophages , 2003, Cellular microbiology.

[45]  M. Venkatesan,et al.  Genetic analysis of Shigella sonnei form I antigen: identification of a novel IS630 as an essential element for the form I antigen expression. , 1998, Microbial pathogenesis.

[46]  P. D'Arcy Hart,et al.  Ammonia inhibits phagosome–lysosome fusion in macrophages , 1980, Nature.

[47]  R. Overbeek,et al.  Missing genes in metabolic pathways: a comparative genomics approach. , 2003, Current opinion in chemical biology.

[48]  T. Doak,et al.  A proposed superfamily of transposase genes: transposon-like elements in ciliated protozoa and a common "D35E" motif. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Richard W. Titball,et al.  Tularemia , 1927, Clinical Microbiology Reviews.

[50]  Peter D. Karp,et al.  MetaCyc: a multiorganism database of metabolic pathways and enzymes , 2005, Nucleic Acids Res..

[51]  S C NAGLE,et al.  CHEMICALLY DEFINED MEDIUM FOR THE GROWTH OF PASTEURELLA TULARENSIS , 1960, Journal of bacteriology.

[52]  T. Sicheritz-Pontén,et al.  The genome sequence of Rickettsia prowazekii and the origin of mitochondria , 1998, Nature.