High quality draft genome sequence and description of Occidentia massiliensis gen. nov., sp. nov., a new member of the family Rickettsiaceae
© Mediannikov et al.; licensee BioMed Central Ltd. 2014
Received: 13 June 2014
Accepted: 16 June 2014
Published: 8 December 2014
The family Rickettsiaceae currently includes two genera: Orientia that contains one species, Orientia tsutsugamushi, and Rickettsia that contains 28 species.
Occidentia massiliensis gen. nov., sp. nov. strain OS118T is the type strain of O. massiliensis gen. nov., sp. nov., the type species of the new genus Occidentia gen. nov. within the family Rickettsiaceae. This strain, whose genome is described here, was isolated in France from the soft tick Ornithodoros sonrai collected in Senegal. O. massiliensis is an aerobic, rod-shaped, Gram-negative, obligate intracellular bacillus that may be cultivated in BME/CTVM2 cells.
Here we describe the features of O. massiliensis, together with the complete genomic sequencing and annotation. The 1,469,252 bp long genome (1 chromosome but no plasmid) contains 1,670 protein-coding and 41 RNA genes, including one rRNA operon.
Occidentia massiliensis gen. nov., sp. nov. strain OS18T is the type strain of O. massiliensis gen. nov., sp. nov. This bacterium was isolated from an Ornithodoros sonrai tick collected in Senegal. It is an aerobic, rod-shaped, Gram-negative, obligate intracellular bacillus.
Here we present a summary classification and a set of features for O. massiliensis gen. nov., sp. nov., strain OS18T (CSUR = P764, DSM = 24860) together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the genus Occidentia and its type species, O. massiliensis within the Rickettsiaceae family.
Classification and general features of Occidentia massiliensis strain OS18 T according to the MIGS recommendations 
Evidence code a
Species Occidentia massiliensis
Type strain OS18T
Sample collection time
0.5 m below surface
45 m above sea level
Five other morphologically and genetically indistinguishable isolates were recovered from five other O. sonrai ticks from the same batch. The 16S rRNA nucleotide sequence (GenBank accession number GU937608) of Occidentia massiliensis strain OS18T was 93.7% similar to Orientia tsutsugamushi strain Gilliam (GenBank accession number D38622), the phylogenetically closest species, but formed a separate, well-supported (bootstrap value 98%) sister branch to the O. tsutsugamushi species (Figure 1). This value was lower than the 95% 16S rRNA gene sequence threshold recommended by Stackebrandt and Elbers to delineate a new genus without carrying out DNA-DNA hybridization .
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
April 15, 2013
Biodiversity of the Ornithodoros sonrai tick microbial flora
Growth conditions and DNA isolation
O. massiliensis gen. nov., sp. nov., strain OS18T (CSUR = P764, DSM = 24860) was grown aerobically in BME/CTVM2 cell line at 28°C. Infected cells were harvested from 20 culture flasks. Bacterial purification using a renografin gradient was performed as previously described . A total of 200 μL of bacterial suspension was diluted in 1 ml TE buffer for lysis treatment. After incubation with 2.5 μg/μL lysozyme for 30 minutes at 37°C, the lysis was performed with 1% laurylsarcosyl and 50 μg/μL RNAse A for 1 hr at 37°C, followed by an overnight incubation at 37°C with proteinase K. The DNA was purified three times by phenol-chloroform extraction and then precipitated by addition of ethanol at -20°C overnight. After centrifugation, the DNA was resuspended in 199 μL TE buffer. The DNA concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios-Tecan fluorometer at 69.12 ng/μl.
Genome sequencing and assembly
A 3 kb paired-end sequencing strategy (454 GS FLX Titanium, Roche) was selected. DNA (5 μg) was mechanically fragmented on the Covaris device (KBioScience-LGC Genomics, Teddington, UK) through miniTube-Red 5 kb 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 average size of 3.2 kb. Circularization and nebulization were performed and generated a pattern with an average size of 580 bp. After PCR amplification over 17 cycles followed by double size selection, the single-stranded paired-end library was then quantified on the Genios-Tecan fluorometer with the Quant-iT ribogreen (Invitrogen) at 1,120 pg/μL. The library concentration equivalence was calculated as 3.55 × 109 molecules/μL. The library was stored at -20°C until further use.
The library was clonally amplified with 0.7 cpb in 4 emPCR reactions, with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yield was calculated at 9.16%, within the recommended yield range of between 5 and 20% from the Roche procedure. After amplification, 790,000 beads from the emPCR reaction 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 103,355 passed filter wells were obtained and generated 34.1 Mb of DNA sequence with an average read length of 330 bp.
The passed filter sequences were assembled using Newbler with 90% identity and 40 bp for overlap requirements. The final assembly identified 18 scaffolds and 47 large contigs (>1.5 kb) generating a genome size of 1,47 Mb which corresponds to a coverage of 23.2×.
Nucleotide content and gene count levels of the genome
% of total a
DNA G + C content (bp)
Protein with predicted function (COGs + NR)
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
Bacterial genomes used for the genomic comparison
Genome Size (Mb)
G + C content
O. massiliensis strain OS18T
O. tsutsugamushi strain Boryong
O. tsutsugamushi strain Ikeda
R. bellii strain RML369-C
R. prowazekii strain Madrid E
Genomic comparison of O. massiliensis and other members of the family Rickettsiaceae
Genomic Comparison of O. massiliensis strain OS18 T with other members of the family Rickettsiaceae
O. tsutsugamushi Boryong
O. tsutsugamushi Ikeda
O. tsutsugamushi Boryong
O. tsutsugamushi Ikeda
Strain OS18 shares a maximum 93.76% 16S rRNA identity with O. tsutsugamushi, its closest phylogenetic neighbor, and 91.61% with R. prowazekii. These values are lower than the 95% threshold proposed by Stackebrandt and Ebers to delineate genera . In addition, the genomic comparison of O. massiliensis and members of two genera from the family Rickettsiaceae demonstrated that the former species exhibits AGIOS values similar to those obtained by comparison of genera, but much lower than those obtained by intra-genus strain comparison.
Moreover, the morphological (monotrichous flagellum, weak coloration by Gimenez staining, concentration around the mitochondria inside infected cell, large periplasmatic space) and epidemiological (association with soft ticks, inability to grow in L929 and XTC cell lines) evidence also differentiates strain OS18 from other members of the family Rickettsiaceae.
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Occidentia massiliensis gen. nov., sp. nov., that contains strain OS18T. This bacterium has been isolated in France from a tick collected in Senegal.
Description of Occidentia gen. nov
Occidentia (oc.ci.den’tia N.L. fem. Adj. occidentia, of the occident, for the western part of Africa where the tick from which the type strain was isolated, was collected, and in contrast with Orientia, the name of its phylogenetically closest relative, distributed in Asia).
Gimenez positive and weakly Gram-negative rods. Strictly intracellular. Non-spore-forming. Grows in BME/CTVM2 tick cells at 28°C. The bacteria multiply freely in the cytoplasm, but not the nucleus, of host cells. Monotrichous flagellum. Habitat: Ornithodoros sonrai. Type species: Occidentia massiliensis.
Description of Occidentia massiliensis gen. nov., sp. nov.
Occidentia massiliensis (mas.si.li.en’sis. L. gen. fem. n. massiliensis, of Massilia, the Latin name of Marseille, France, where strain OS18T was first grown, identified and characterized).
Gimenez positive and weakly Gram-negative rods. Strictly intracellular. Non-spore-forming. Grows in BME/CTVM2 tick cells at 28°C. The bacteria multiply freely in the cytoplasm, but not the nucleus, of host cells. Monotrichous flagellum. The mean length and width of the bacteria are 1.23 ± 0.19 μm and 0.42 ± 0.06 μm, respectively. Bacteria exhibit a large periplasmic space of 0.028 ± 0.007 μm. The genome is 1,469,252-bp long and contains 1,502 protein-coding and 41 RNA genes. The 16S rRNA and genomic sequences are deposited in GenBank under accession numbers GU937608 and CANJ00000000, respectively. The genomic G + C content is 29.05%. The type strain OS18T (CSUR = P764, DSM = 24860) was isolated from an Ornithodoros sonrai soft tick collected in Senegal.
We are grateful to Marie-Laure Birg, Nathalie Duclos, Audrey Borg and Denis Pyak for their technical help. The present work was funded 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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