Genome Sequence of Rickettsia bellii Illuminates the Role of Amoebae in Gene Exchanges between Intracellular Pathogens

The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair–long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic “melting pot” where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.

[1]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[2]  S. Makino,et al.  Bacterial genomic reorganization upon DNA replication. , 2001, Science.

[3]  M. R. Brown,et al.  Trojan horses of the microbial world: protozoa and the survival of bacterial pathogens in the environment. , 1994, Microbiology.

[4]  S. Salzberg,et al.  Evidence for symmetric chromosomal inversions around the replication origin in bacteria , 2000, Genome Biology.

[5]  D. Raoult,et al.  Phylogeny of Rickettsia spp. inferred by comparing sequences of 'gene D', which encodes an intracytoplasmic protein. , 2001, International journal of systematic and evolutionary microbiology.

[6]  D. Raoult,et al.  Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB). , 2000, International journal of systematic and evolutionary microbiology.

[7]  J. Adachi,et al.  MOLPHY version 2.3 : programs for molecular phylogenetics based on maximum likelihood , 1996 .

[8]  C. Fuqua,et al.  The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes , 1996, Journal of bacteriology.

[9]  P. Cossart,et al.  Actin-based motility of intracellular pathogens. , 2005, Current opinion in microbiology.

[10]  D. E. Bradley,et al.  Specification of surface mating systems among conjugative drug resistance plasmids in Escherichia coli K-12 , 1980, Journal of bacteriology.

[11]  M. Wagner,et al.  Bacterial Endosymbionts of Free-living Amoebae1 , 2004, The Journal of eukaryotic microbiology.

[12]  S. Eddy.  Hidden Markov models. , 1996, Current opinion in structural biology.

[13]  T. Rowbotham.  Isolation of Legionella pneumophila from clinical specimens via amoebae, and the interaction of those and other isolates with amoebae. , 1983, Journal of clinical pathology.

[14]  Michael Wagner,et al.  Amoebae as Training Grounds for Intracellular Bacterial Pathogens , 2005, Applied and Environmental Microbiology.

[15]  O. Harb,et al.  From protozoa to mammalian cells: a new paradigm in the life cycle of intracellular bacterial pathogens. , 2000, Environmental microbiology.

[16]  W. Burgdorfer,et al.  Rickettsia bellii sp. nov.: a Tick-Borne Rickettsia, Widely Distributed in the United States, That Is Distinct from the Spotted Fever and Typhus Biogroups , 1983 .

[17]  L. Frost,et al.  F factor conjugation is a true type IV secretion system. , 2003, FEMS microbiology letters.

[18]  Temple F. Smith,et al.  Patterns of Genome Organization in Bacteria , 1998, Science.

[19]  D. Raoult,et al.  Rickettsioses as paradigms of new or emerging infectious diseases , 1997, Clinical microbiology reviews.

[20]  R. Heinzen,et al.  Dynamics of Actin-Based Movement byRickettsia rickettsii in Vero Cells , 1999, Infection and Immunity.

[21]  D. Raoult,et al.  History of the ADP/ATP-Translocase-Encoding Gene, a Parasitism Gene Transferred from a Chlamydiales Ancestor to Plants 1 Billion Years Ago , 2004, Applied and Environmental Microbiology.

[22]  H. Goodson,et al.  Actin and ARPs: action in the nucleus. , 2004, Trends in cell biology.

[23]  Gilbert Greub,et al.  A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system , 2004, BMC Microbiology.

[24]  D Raoult,et al.  Selfish DNA in protein-coding genes of Rickettsia. , 2000, Science.

[25]  M. Wagner,et al.  ATP/ADP Translocases: a Common Feature of Obligate Intracellular Amoebal Symbionts Related to Chlamydiae and Rickettsiae , 2004, Journal of bacteriology.

[26]  R. Kahn,et al.  A Bacterial Guanine Nucleotide Exchange Factor Activates ARF on Legionella Phagosomes , 2002, Science.

[27]  J. Weissenbach,et al.  Mechanisms of Evolution in Rickettsia conorii and R. prowazekii , 2001, Science.

[28]  D. Higgins,et al.  T-Coffee: A novel method for fast and accurate multiple sequence alignment. , 2000, Journal of molecular biology.

[29]  Sudhir Kumar,et al.  MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment , 2004, Briefings Bioinform..

[30]  M. W. Taylor,et al.  'Candidatus Protochlamydia amoebophila', an endosymbiont of Acanthamoeba spp. , 2005, International journal of systematic and evolutionary microbiology.

[31]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.

[32]  L. Frost,et al.  Analysis of the sequence and gene products of the transfer region of the F sex factor , 1994, Microbiological reviews.

[33]  Peer Bork,et al.  SMART: identification and annotation of domains from signalling and extracellular protein sequences , 1999, Nucleic Acids Res..

[34]  Josephine C. Adams,et al.  The RickA protein of Rickettsia conorii activates the Arp2/3 complex , 2004, Nature.

[35]  Dmitrij Frishman,et al.  Illuminating the Evolutionary History of Chlamydiae , 2004, Science.

[36]  K. Gerdes,et al.  Prokaryotic toxin–antitoxin stress response loci , 2005, Nature Reviews Microbiology.

[37]  Cathy H. Wu,et al.  UniProt: the Universal Protein knowledgebase , 2004, Nucleic Acids Res..

[38]  E V Koonin,et al.  Rickettsiae and Chlamydiae: evidence of horizontal gene transfer and gene exchange. , 1999, Trends in genetics : TIG.

[39]  S. B. Horowitz,et al.  Nucleocytoplasmic transport and distribution of an amino acid, in situ. , 1975, Journal of cell science.

[40]  D. Raoult,et al.  Determination of genome sizes of Rickettsia spp. within the spotted fever group, using pulsed-field gel electrophoresis , 1992, Journal of bacteriology.

[41]  C. L. Jackson,et al.  Phylogenetic analysis of Sec7-domain-containing Arf nucleotide exchangers. , 2004, Molecular biology of the cell.

[42]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[43]  Robert C. Edgar,et al.  MUSCLE: a multiple sequence alignment method with reduced time and space complexity , 2004, BMC Bioinformatics.

[44]  John B. Anderson,et al.  CDD: a Conserved Domain Database for protein classification , 2004, Nucleic Acids Res..

[45]  O. Gascuel,et al.  Theoretical foundation of the balanced minimum evolution method of phylogenetic inference and its relationship to weighted least-squares tree fitting. , 2003, Molecular biology and evolution.

[46]  K. Schleifer,et al.  In Situ Detection of Novel Bacterial Endosymbionts of Acanthamoeba spp. Phylogenetically Related to Members of the Order Rickettsiales , 1999, Applied and Environmental Microbiology.

[47]  S. Eddy,et al.  tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. , 1997, Nucleic acids research.

[48]  GnanaSundar Rajendiran,et al.  Clustering Method for Repeat Analysis in DNA sequences , 2008 .

[49]  Gilbert Greub,et al.  Microorganisms Resistant to Free-Living Amoebae , 2004, Clinical Microbiology Reviews.

[50]  L. C. Moore,et al.  Nuclear envelope permeability , 1975, Nature.

[51]  D. Raoult,et al.  Phylogenetic analysis of spotted fever group rickettsiae by study of the outer surface protein rOmpA. , 1998, International journal of systematic bacteriology.

[52]  J. Claverie,et al.  The Genome Sequence of Rickettsia felis Identifies the First Putative Conjugative Plasmid in an Obligate Intracellular Parasite , 2005, PLoS biology.

[53]  Jean-Michel Claverie,et al.  Protein coding palindromes are a unique but recurrent feature in Rickettsia. , 2002, Genome research.

[54]  P. Fuerst,et al.  Ancestral divergence of Rickettsia bellii from the spotted fever and typhus groups of Rickettsia and antiquity of the genus Rickettsia. , 1994, International journal of systematic bacteriology.

[55]  K. Gerdes,et al.  Toxin–antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes , 2005, Nucleic acids research.

[56]  P. Cossart,et al.  Ku70, a Component of DNA-Dependent Protein Kinase, Is a Mammalian Receptor for Rickettsia conorii , 2005, Cell.