Non-contiguous finished genome sequence and description of Bartonella senegalensis sp. nov.
© The Author(s) 2013
Published: 15 June 2013
Bartonella senegalensis sp. nov. strain OS02T is the type strain of B. senegalensis sp. nov., a new species within the genus Bartonella. This strain, whose genome is described here, was isolated in Senegal from the soft tick Ornithodoros sonrai, the vector of relapsing fever. B. senegalensis is an aerobic, rod-shaped, Gram-negative bacterium. Here we describe the features of this organism, together with the complete genome sequence and its annotation. The 1,966,996 bp-long genome contains 1,710 protein-coding and 46 RNA genes, including 6 rRNA genes.
Bartonella is the only genus of the family Bartonellaceae of Alphaproteobacteria. To date, 29 Bartonella species have been validly published [1,2], and many isolates have yet to be described. These bacteria are facultative intracellular pathogens, many of which infect erythrocytes . At least 13 Bartonella species are associated with human diseases. B. bacilliformis, B. quintana and B. henselae, are relatively common human pathogens and cause Carrión’s disease, trench fever and cat scratch fever, respectively. Different species of Bartonella are also associated with chronic bacteremia and/or endocarditis, bacillary angiomatosis, peliosis hepatitis, retinitis, uveitis, and myocarditis .
The epidemiological cycle of bartonellae consists of a reservoir host, which is a vertebrate with a chronic intravascular infection and sustained bacteremia, and a vector (usually a blood-sucking arthropod such as fleas, sandflies or lice) that transfers the bacteria from the reservoir to a susceptible host. Bartonella species are typically associated with a specific primary host; e.g., B. henselae is commonly found in domestic and wild felids all over the world, including Africa [5–7], whereas B. bacilliformis is human-specific. Animal hosts of bartonellae include dogs, rabbits, coyotes, foxes, cattle, deer, elk and multiple rodent species [6,8–10]. For most pathogenic bartonellae (except B. bacilliformis and B. quintana), humans are accidental (secondary) hosts .
In 2003, La Scola et al. proposed a multilocus sequence analysis based on 4 genes and one intergenic spacer as a tool for the description of new Bartonella species . Among these genetic markers, two, i.e., gltA and rpoB, were particularly discriminatory, with new Bartonella isolates considered as new species if they exhibit <96.0% and <95.4% sequence identity with other validly published species for the 327- and 825-bp fragments of the gltA and rpoB genes, respectively [2,11–13]. This strategy of combining sequences from several genes, usually housekeeping genes, is congruent with the “gold-standard” DNA-DNA reassociation for several bacterial genera .
In this study, we used La Scola’s criteria and described the genome sequence as well as main phenotypic characteristics of strain OS02T. Here, we present a summary classification and a set of features for B. senegalensis sp. nov. strain OS02T together with the description of the complete genomic sequence and annotation. These characteristics support the definition of the species B. senegalensis.
Classification and features
Classification and general features of Bartonella senegalensis strain OS02T.
Species Bartonella senegalensis
Type strain OS02T
Growth in BHI medium + 5% NaCl
Soft tick Ornithodoros sonrai
Sample collection time
~ 0.5 m under surface
45 m above sea level
Strain OS02T exhibited neither catalase nor oxidase activity. Biochemical characteristics were assessed using an Anaerobe Identification Test Panel AN MicroPlate™ (Biolog Inc., Hayward, CA, USA). None of 95 biochemical tests available (including D-mannose, D-fructose and D-galactose) were positive. Similar profiles were previously observed for other Bartonella species .
Genome sequencing information
Genome project history
One paired-end 3-kb library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
Genbank Date of Release
August 17, 2012
Biodiversity of Ornithodoros sonrai flora
Growth conditions and DNA isolation
B. senegalensis sp. nov. strain OS02T (DSM 23168; CSUR B623) was grown on 5% sheep blood-enriched Columbia agar at 37°C in a 5% CO2 atmosphere. Four Petri dishes were spread and resuspended in 3×100 µl of G2 buffer (EZ1 DNA Tissue kit, Qiagen). A first mechanical lysis was performed by glass powder on the Fastprep-24 device (Sample Preparation system; MP Biomedicals, USA) using 2×20-second cycles. DNA was then treated with 2.5 µg/µL lysozyme (30 minutes at 37°C) and extracted through the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was then concentrated and purified on a Qiamp kit (Qiagen). The yield and concentration were measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 130.4 ng/µl.
Genome sequencing and assembly
DNA (5 µg) was mechanically fragmented on a Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size of 3–4 kb. The DNA fragmentation was visualized using the Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 3.475 kb. The library was constructed according to the 454 GS FLX Titanium paired-end protocol. Circularization and nebulization were performed and generated a pattern with an optimum at 641 bp. After PCR amplification over 17 cycles followed by double size selection, the single-stranded paired-end library was then quantified with the BioAnalyzer on a DNA labchip RNA pico 6,000 at 323 pg/µL. The library concentration equivalence was calculated as 9.24E+08 molecules/µL. The library was stored at −20°C until further use.
The library was clonally amplified with 1 cpb and 1.5 cpb in 4 and 3 emPCR reactions, respectively, with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the 1 cpb and 1.5 cpb emPCR were determined to be 3.08% and 8%, respectively. After amplification, 790,000 beads from the 2 emPCR conditions were loaded on a ¼ region on the GS Titanium PicoTiterPlate PTP Kit 70×75 and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche). The run was analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). A total of 200,243 passed filter wells were obtained and generated 57.62 Mb of DNA sequence with an average length of 287 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp for overlap requirements. The final assembly identified 9 scaffolds and 63 large contigs (≥1,500 bp), generating a genome size of 1.98 Mb, which corresponds to 29.10× equivalent genome.
Open reading frames (ORFs) were predicted using PRODIGAL  with default parameters, but predicted ORFs were excluded if they spanned a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank database  using BLASTP and the Clusters of Orthologous Groups (COG) database using COGNITOR . The prediction of RNA genes, i.e., rRNAs, tRNAs and other RNAs, was performed using the RNAmmer  and ARAGORN  algorithms. The transmembrane helices and signal peptides were identified using TMHMM  and SignalP , respectively.
Nucleotide content and percentage of the genome
% of totala
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Protein with predicted function
Genes assigned to COG
Genes with peptide signal
Genes with transmembrane helices (≥3)
Number of genes associated with the 25 general COG functional categories†.
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover and chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Insights from the genome sequence
Compared to B. henselae strain Houston (GenBank accession number NC_005956), its closest phylogenetic neighbor, B. senegalensis strain OS02T had a larger genome (1,966,996 and 1,931,047 bp, respectively), more genes (1,756 and 1,491 genes, respectively) and a higher G+C content (38.6 and 38%, respectively). The protein-coding genes present in B. senegalensis but absent or split in B. henselae included multidrug-efflux transporter, membrane protein formate-tetrahydrofolate ligase, formate-tetrahydrofolate ligase, glycoside hydrolase family 3-like, glycoside hydrolase family 3-like, putative major facilitator superfamily, SAM-dependent methyltransferases, resolvases, toxin-antitoxin modules, transposases, ubiquinol-cytochrome C reductase, LeuA2, and phage proteins, as well as several hypothetical proteins.
On the basis of phylogenetic and genotypic analyses, we formally propose the creation of Bartonella senegalensis sp. nov., which contains strain OS02T. This bacterium was isolated in Senegal.
Description of Bartonella senegalensis sp. nov.
Bartonella senegalensis (se.ne.ga.len′sis. N.L. fem. adj. senegalensis referring to Senegal, the African country that is home to the Ornithodoros sonrai tick from which the type strain was isolated).
Colonies are opaque, grey and 0.5 to 1.0 mm in diameter on blood-enriched Columbia agar. Cells are rod-shaped without flagellae. Length and width are 1,254.4±329.3 nm and 533.3±100.5 nm, respectively. Growth is only obtained at 37°C. Cells stain Gram-negative, are non-endospore-forming, and are non-motile. Catalase and oxidase activities are absent. No biochemical activity is observed using the Anaerobe Identification Test Panel AN MicroPlate.
The ITS, 16S rRNA, ftsZ, rpoB and gltA genes, and draft genome sequences are deposited in GenBank under accession numbers HM636451, HM636442, HM636445, HM636454, HM636448 and CALV00000000, respectively. The genome is 1,966,996 bp long and contains 1,710 protein-coding and 46 RNA genes, including 6 rRNA genes. The G+C content is 38.6%. The type strain OS02T (DSM 23168, CSUR B623) was isolated from an O. sonrai soft tick collected in a rodent burrow in a rural village in Senegal.
We are grateful to Denis Pyak, Audrey Borg, and Geetha Subramanian for their technical help. The present work was partly supported by the Agence Nationale de Recherche grant 2010 MALEMAF (research on emergent pathogens in Africa) and the Mediterranée Infection Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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