The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number.

Heartwater, a tick-borne disease of domestic and wild ruminants, is caused by the intracellular rickettsia Ehrlichia ruminantium (previously known as Cowdria ruminantium). It is a major constraint to livestock production throughout subSaharan Africa, and it threatens to invade the Americas, yet there is no immediate prospect of an effective vaccine. A shotgun genome sequencing project was undertaken in the expectation that access to the complete protein coding repertoire of the organism will facilitate the search for vaccine candidate genes. We report here the complete 1,516,355-bp sequence of the type strain, the stock derived from the South African Welgevonden isolate. Only 62% of the genome is predicted to be coding sequence, encoding 888 proteins and 41 stable RNA species. The most striking feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number, which contributes to the low proportion of coding sequence. These repeats have mediated numerous translocation and inversion events that have resulted in the duplication and truncation of some genes and have also given rise to new genes. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Rather then being the result of the reductive evolution seen in other intracellular bacteria, these pseudogenes appear to be the product of ongoing sequence duplication events.

[1]  J. Bezuidenhout,et al.  The historical background and global importance of heartwater. , 1987, The Onderstepoort journal of veterinary research.

[2]  C. Claudel-Renard,et al.  Enzyme-specific profiles for genome annotation: PRIAM. , 2003, Nucleic acids research.

[3]  P. Bork.  Hundreds of ankyrin‐like repeats in functionally diverse proteins: Mobile modules that cross phyla horizontally? , 1993, Proteins.

[4]  E. Zweygarth,et al.  Amino Acid Content of Cell Cultures Infected with Cowdria ruminantium Propagated in a Protein‐free Medium , 2002, Annals of the New York Academy of Sciences.

[5]  G. Pazour,et al.  Delineation of the regulatory region sequences of Agrobacterium tumefaciens virB operon. , 1989, Nucleic acids research.

[6]  D. Sankoff,et al.  An ancestral mitochondrial DNA resembling a eubacterial genome in miniature , 1997, Nature.

[7]  Y. Rikihisa,et al.  Cloning and Characterization of Multigenes Encoding the Immunodominant 30-Kilodalton Major Outer Membrane Proteins ofEhrlichia canis and Application of the Recombinant Protein for Serodiagnosis , 1998, Journal of Clinical Microbiology.

[8]  Ling V. Sun,et al.  Phylogenomics of the Reproductive Parasite Wolbachia pipientis wMel: A Streamlined Genome Overrun by Mobile Genetic Elements , 2004, PLoS biology.

[9]  E. Rocha.  An appraisal of the potential for illegitimate recombination in bacterial genomes and its consequences: from duplications to genome reduction. , 2003, Genome research.

[10]  Diarmaid Hughes,et al.  Evaluating genome dynamics: the constraints on rearrangements within bacterial genomes , 2000, Genome Biology.

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

[12]  G. Gutman,et al.  Slipped-strand mispairing: a major mechanism for DNA sequence evolution. , 1987, Molecular biology and evolution.

[13]  C. Kurland,et al.  Reductive evolution of resident genomes. , 1998, Trends in microbiology.

[14]  Mikhail S. Gelfand,et al.  Combining diverse evidence for gene recognition in completely sequenced bacterial genomes , 1998, German Conference on Bioinformatics.

[15]  Kim Rutherford,et al.  Artemis: sequence visualization and annotation , 2000, Bioinform..

[16]  Peter Kuhn,et al.  Mechanistic studies of a flavin-dependent thymidylate synthase. , 2004, Biochemistry.

[17]  M. de Groot,et al.  Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein. , 1991, Veterinary microbiology.

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

[19]  G. Benson,et al.  Tandem repeats finder: a program to analyze DNA sequences. , 1999, Nucleic acids research.

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

[21]  B. Barrell,et al.  The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences , 2000, Nature.

[22]  Ning Zhi,et al.  Immunodominant Major Outer Membrane Proteins ofEhrlichia chaffeensis Are Encoded by a Polymorphic Multigene Family , 1998, Infection and Immunity.

[23]  E. D. de Villiers,et al.  Genome size and genetic map of Cowdria ruminantium. , 2000, Microbiology.

[24]  J. Plessis.  A METHOD FOR DETERMINING THE COWDRIA RUMINANTIUM INFECTION RATE OF AMBLYOMMA HEBRAEUM: EFFECTS IN MICE INJECTED WITH TICK HOMOGENATES , 1985 .

[25]  S. Acinas,et al.  Divergence and Redundancy of 16S rRNA Sequences in Genomes with Multiple rrn Operons , 2004, Journal of bacteriology.

[26]  P. Hooykaas,et al.  Analysis of the complete nucleotide sequence of the Agrobacterium tumefaciens virB operon. , 1988, Nucleic acids research.

[27]  Y. Rikihisa,et al.  Multiple p44 Genes Encoding Major Outer Membrane Proteins Are Expressed in the Human Granulocytic Ehrlichiosis Agent* , 1999, The Journal of Biological Chemistry.

[28]  Rainer Merkl,et al.  SIGI: score-based identification of genomic islands , 2004, BMC Bioinformatics.

[29]  H. Steyn,et al.  Phylogenetic Relationships among Ehrlichia ruminantium Isolates , 2003, Annals of the New York Academy of Sciences.

[30]  M. Dehio,et al.  The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells , 2004, Molecular microbiology.

[31]  Y. Rikihisa,et al.  Characterization and Transcriptional Analysis of Gene Clusters for a Type IV Secretion Machinery in Human Granulocytic and Monocytic Ehrlichiosis Agents , 2002, Infection and Immunity.

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

[33]  Gregory Kucherov,et al.  mreps: efficient and flexible detection of tandem repeats in DNA , 2003, Nucleic Acids Res..

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

[35]  Søren Brunak,et al.  A Neural Network Method for Identification of Prokaryotic and Eukaryotic Signal Peptides and Prediction of their Cleavage Sites , 1997, Int. J. Neural Syst..

[36]  Amos Bairoch,et al.  PROSITE: A Documented Database Using Patterns and Profiles as Motif Descriptors , 2002, Briefings Bioinform..

[37]  C. Kurland,et al.  Molecular phylogeny and rearrangement of rRNA genes in Rickettsia species. , 1999, Molecular biology and evolution.

[38]  J. Bonfield,et al.  A new DNA sequence assembly program. , 1995, Nucleic acids research.

[39]  P. Christie,et al.  Type IV secretion: intercellular transfer of macromolecules by systems ancestrally related to conjugation machines , 2001, Molecular microbiology.

[40]  Fasheng Zhang,et al.  Computational differentiation of N-terminal signal peptides and transmembrane helices. , 2003, Biochemical and biophysical research communications.

[41]  B. Simbi,et al.  A subset of Cowdria ruminantium genes important for immune recognition and protection. , 2001, Gene.

[42]  Kelly A Brayton,et al.  Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[43]  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 , 2003, Applied and Environmental Microbiology.

[44]  K. Wise,et al.  Elongated versions of Vlp surface lipoproteins protect Mycoplasma hyorhinis escape variants from growth-inhibiting host antibodies , 1997, Infection and immunity.

[45]  W R Pearson,et al.  Flexible sequence similarity searching with the FASTA3 program package. , 2000, Methods in molecular biology.

[46]  T. McElwain,et al.  The immunoprotective Anaplasma marginale major surface protein 2 is encoded by a polymorphic multigene family , 1994, Infection and immunity.

[47]  H. van Heerden,et al.  Characterization of a major outer membrane protein multigene family in Ehrlichia ruminantium. , 2004, Gene.

[48]  K. Woodford,et al.  DNA Secondary Structures and the Evolution of Hypervariable Tandem Arrays* , 1997, The Journal of Biological Chemistry.

[49]  H. Yoshikawa,et al.  Genes and their organization in the replication origin region of the bacterial chromosome , 1992, Molecular microbiology.

[50]  R. Doolittle,et al.  Of urfs and orfs , 1986 .

[51]  J. Celli,et al.  Organelle robbery: Brucella interactions with the endoplasmic reticulum. , 2004, Current opinion in microbiology.

[52]  R. G. Hewinson,et al.  Recognition of Mycobacterial Epitopes by T Cells across Mammalian Species and Use of a Program That Predicts Human HLA-DR Binding Peptides To Predict Bovine Epitopes , 2003, Infection and Immunity.

[53]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[54]  M. Borodovsky,et al.  GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. , 2001, Nucleic acids research.

[55]  G. Heijne.  A new method for predicting signal sequence cleavage sites. , 1986 .

[56]  N. Moran,et al.  Genes Lost and Genes Found: Evolution of Bacterial Pathogenesis and Symbiosis , 2001, Science.

[57]  D. Knowles,et al.  Conservation of the unique rickettsial rRNA gene arrangement in Anaplasma. , 2002, International journal of systematic and evolutionary microbiology.

[58]  E. Zweygarth,et al.  Serum‐free Media for the in Vitro Cultivation of Cowdria ruminantium , 1998, Annals of the New York Academy of Sciences.

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

[60]  Y. Rikihisa,et al.  Ehrlichia chaffeensis and Anaplasma phagocytophilum Lack Genes for Lipid A Biosynthesis and Incorporate Cholesterol for Their Survival , 2003, Infection and Immunity.

[61]  M. Riley,et al.  Organization of the bacterial chromosome , 1990, Microbiological reviews.

[62]  E. D. de Villiers,et al.  Construction and initial analysis of a representative lambda ZAPII expression library of the intracellular rickettsia Cowdria ruminantium: cloning of map1 and three other Cowdria genes. , 1997, Veterinary parasitology.

[63]  D. McKeever,et al.  Analysis of T-cell responses in cattle immunized against heartwater by vaccination with killed elementary bodies of Cowdria ruminantium , 1997, Infection and immunity.

[64]  Bezuidenhout Jd,et al.  The production of heartwater vaccine. , 1987 .

[65]  J. Lobry.  Asymmetric substitution patterns in the two DNA strands of bacteria. , 1996, Molecular biology and evolution.