Genome sequence of Coxiella burnetii strain Namibia
- Mathias C Walter†1, 2,
- Caroline Öhrman†3,
- Kerstin Myrtennäs3,
- Andreas Sjödin3,
- Mona Byström3,
- Pär Larsson3,
- Anna Macellaro3,
- Mats Forsman†3 and
- Dimitrios Frangoulidis†4Email author
© Walter et al.; licensee BioMed Central. 2014
Received: 19 May 2014
Accepted: 17 November 2014
Published: 29 December 2014
We present the whole genome sequence and annotation of the Coxiella burnetii strain Namibia. This strain was isolated from an aborting goat in 1991 in Windhoek, Namibia. The plasmid type QpRS was confirmed in our work. Further genomic typing placed the strain into a unique genomic group. The genome sequence is 2,101,438 bp long and contains 1,979 protein-coding and 51 RNA genes, including one rRNA operon. To overcome the poor yield from cell culture systems, an additional DNA enrichment with whole genome amplification (WGA) methods was applied. We describe a bioinformatics pipeline for improved genome assembly including several filters with a special focus on WGA characteristics.
KeywordsCoxiella burnetii Q fever Whole genome sequencing Next generation sequencing (NGS) Assembly Annotation Whole genome amplification
Creation of whole genome information of Coxiella burnetii is a cumbersome procedure. All work with living strains of C. burnetii is impaired by the necessity to handle strains under Biosafety 3 conditions. The enrichment of this bacterium is normally done in animal derived cell culture systems with a peak of replication after 5 to 7 days of growth. The overall yield of bacteria, however, is less than that obtained by “classical” growth of bacteria on artificial media. Alternative enrichment methods, like animal inoculation and cultivation in hen eggs, present various problems and risks in processing, thus are not in common use for C. burnetii. The required amount and quality of DNA for whole genome sequencing of C. burnetii is not easily obtained by cell culture. Furthermore, DNA isolation is not always successful and not all DNA preparations are of a quality suitable for sequencing purposes. To overcome these problems, WGA techniques may present an attractive alternative for generation of C. burnetii DNA . Such assays possess an impressive power to amplify traces of DNA to a satisfactory quantity. However, a careful evaluation with the species of interest is mandatory to judge its suitability. Repeat structures and insertions sequences (IS) in particular might influence the quality of amplification. The Coxiella genome shows IS elements with sometimes more than 100 copies , stressing the importance of thorough evaluation of WGA techniques. We chose a special variant of WGA, the MDA method, that has been successful applied [3, 4] and is commercially available from different companies (RepliG, Qiagen, Hilden, Germany and GenomiPhi, GE Healthcare, Freiburg, Germany) . Very recently the RepliG kit was used with Coxiella DNA and evaluated at 20 selected loci .
In this study, we describe a method for obtaining high quality DNA from Coxiella suitable for whole genome sequencing. We also evaluate the utility of WGA for Coxiella whole genome sequencing and WGA induced demands on downstream bioinformatics processing of sequence data, especially for genome assembly and finishing. The whole genome sequence presented here is the first of a C. burnetii strain originating from the African continent and will increase the genomic knowledge for this region.
C. burnetii is the causative pathogen of the zoonotic disease Q fever, which has a worldwide distribution with the only exceptions of New Zealand and Antarctica. The bacterium was first independently described and isolated in Australia and the United States of America in 1937 [7, 8]. C. burnetii is an obligately intracellular, small, Gram-negative, non-motile, pleomorphic, coccobacillary bacterium (0.2 – 0.4 μm × 0.4 – 1 μm). Atypically, its Gram-negative membrane cannot be stained using Gram techniques, but can be visualized by the Gimenez method .
Classification and general features of C. burnetii strain Namibia according to the MIGS recommendations 
Evidence code a
Species: Coxiella burnetii
35 – 37°C
pH range; Optimum
intracellular, polyhostal long persistence in the environment
In its development cycle, C. burnetii generates both large (LCV) and small cell variants (SCV). The latter are more environmentally stable and present the infectious particles incorporated by different hosts. After uptake by macrophages, the LCV is formed within phagolysosomes.
The bacterium exists in two antigenic phases, which are analogous to the smooth (phase I) and rough (phase II) LPS forms seen among the Enterobacteriaceae. Phase I bacteria can be observed during natural infections of humans and animals, whereas bacteria in phase II, which are mainly non-virulent, evolve after several passages in embryonated hen eggs or cell cultures. Transitions between both forms have been described .
C. burnetii has a large reservoir of hosts including many wild and domestic mammals, birds, reptiles, fish and even arthropods such as ticks and flies. Due to its transmission by inhalation, low infectious dose, high stability, and prior weaponization, C. burnetii is classified as a category B agent of bioterrorism by the Centers for Disease Control (CDC, Atlanta, USA) . Epidemiological studies have demonstrated that the most frequent route of human C. burnetii infections is via domestic ruminants such as sheep, goats, or cattle. These animals may be chronically infected without showing any clinical symptoms and shed vast numbers of the bacterium into the environment, mainly during parturition. Counts of C. burnetii in excess of 109 bacteria per gram have been recorded in placental tissue, but in other birth-associated products such as amniotic fluids or in milk, high quantities of C. burnetii may also be present. Particularly high counts have been obtained from tick feces with reports of 1010 living organisms per gram . Despite this, ticks do not appear to be a significant risk factor for acquisition of human infection .
The organism is a highly infectious agent, with experimental estimates suggesting an infectious dose of less than 10 organisms for manifestation of an infection . Furthermore, coxiellae are highly resistant to both heat and desiccation, ubiquitously available, and their aerosolized state is infectious over several kilometers [16, 17].
C. burnetii strains appear with five different plasmid types, four different plasmids (QpH1, QpRS, QpDV, and QpDG) and one type with a chromosomal plasmid-homologous sequence [18–22]. The characterization of these plasmids led to a classification into five genomic groups. Some plasmid types could be associated with various geographic regions. A formerly hypothesized correlation of these genomic groups with virulence or clinical manifestation could not be confirmed in later studies .
Genome sequencing information
Genome project history
Improved high-quality draft
Nextera DNA Sample Prep Kit
Illumina MiSeq, 2x 150 paired-end
Gene calling method
Prodigal, GeneMarkS, Glimmer
NCBI Taxonomy ID
Genbank Date of Release
October 16, 2014
Medical, bioforensic, evolution
Source Material Identifier
Growth conditions and DNA isolation
BGM cells (Flow Laboratories, Rockville, MD, USA) were grown in Eagles minimal essential medium (MEM) supplemented with Earls salts, 2 mM L-glutamax, 5% Fetal Calf Serum (FCS), 1% Non-Essential Amino acids (NEA) and 0.2% sodium bicarbonate (Sigma-Aldrich, St Louis, MO, USA). Confluent cell layers were infected with bacteria and incubated at 37°C. Fresh media was added after 20–24 h. To enhance vacuole formation, the infected confluent BGM cells were divided using trypsin. C. burnetii cells were collected from the medium of actively growing cultures after 7–8 days by differential centrifugation. An initial centrifugation step to remove cell debris was performed at 500 × g (1,500 rpm) for 5 minutes at 4°C, followed by a second centrifugation step to collect the bacteria at 2,550 × g (3,500 rpm) for one hour at 4°C.
The bacteria from confluently growing cell cultures were harvested by differential centrifugation as described above. The bacteria were washed twice in PBS. The bacterial pellet was then resuspended in 50 mM Tris (pH 7.8) and mixed with 10 mM MgSO4 solution containing 20 μg DNase (Ambion, Life Technologies, Carlsbad, CA, USA). The resulting suspension was incubated at 37°C for 30 minutes. 0.5% SDS and 50 μg/ml proteinase K solution were then added and the sample was incubated at 56°C for one hour. After cooling to room temperature, 100 mM Tris (pH 7.8), 1 mM EDTA, a 15% sucrose solution, and 1 mg/ml lysozyme solution (Sigma-Aldrich) were added and the resulting mixture was incubated at 37°C for 16 h. On the following day, 100 mM Tris (pH 12.0), 1 mM EDTA, and 5% SDS were added and the sample was incubated at 56°C for one hour.
The sample was then cooled to room temperature and treated with phenol/chloroform twice before three volumes of ice cold (−20°C) 99.5% ethanol to precipitate the DNA were added. After incubation at −20°C for 30 min, the sample was centrifuged at 19,000 × g (15,000 rpm) for 30 min. The pellet was then resuspended in 1 × TE containing 50 μg RNase (Epicentre, Madison, WI, USA) and incubated at 37°C for one hour. Proteinase K (500 μg, Epicentre) was then added and the resulting mixture was incubated for another hour at 37°C. The sample was treated with phenol/chloroform twice before precipitation of the DNA by adding 0.1 volume of 3 M sodium acetate and 2.5 volumes of ice cold (−20°C) 99.5% ethanol. The resulting mixture was then incubated at −20°C for 30 min, centrifuged, and washed twice with 80% ethanol. After centrifugation the pellet was air dried and resuspended in 1 × TE.
To obtain larger quantities of DNA for whole genome sequencing, the sample was amplified using the MDA kit Illustra GenomePhi V2 Amplification Kit (GE Healthcare Life Sciences and the REPLI-g UltraFast Mini Kit (Qiagen, Hilden, Germany), respectively, according to the manufacturers’ instructions. Once the amplification was complete, the enzymes were inactivated by heating the sample to 65°C. The product was then diluted with sterile distilled water, the DNA was extracted using phenol/chloroform, and the product was precipitated using 0.1 volumes of 3 M sodium acetate and 2.5 volumes of ice cold (−20°C) 99.5% ethanol followed by incubation for 16 h at −20°C. The precipitated sample was centrifuged at 19,000 × g (15,000 rpm) for 30 minutes at 4°C, washed once with 70% ethanol and air dried. The pellet was then dissolved in 1 × TE and the DNA concentration was estimated using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA).
Genome sequencing and assembly
The isolated DNA was prepared using the Nextera DNA Sample Prep Kit (Illumina, Hayward, CA, USA) and paired-end reads of 150 bp were sequenced on a MiSeq benchtop sequencer (Illumina) at the Swedish Defence Research Agency and according to the manufacturer’s instructions.
The sequenced reads were filtered against the draft genome assembly of Chlorocebus sabaeus (assembly AQIB01), Macaca mulatta (assembly AANU01), and Papio anubis (assembly AHZZ01) as well as four bacterial contaminants: Escherichia coli str. K-12 substr. MG1655 (NC_000913), Mycoplasma arginini 7264 (NZ_AORG01000000), Propionibacterium acnes 6609 (NC_017535) and Streptococcus suis SC84 (NC_012924) using mirabait, a kmer-based read mapping tool . Afterwards, bacterial contaminant reads were blasted against the C. burnetii RefSeq genomes and the contaminant genomes. Reads which are more similar in their full length to C. burnetii were reintegrated into the filtered read set. This step was done as a quality control not to filter too many reads, such as reads mapping to orthologous genes or the ribosomal RNA operon.
Afterwards we used MIRA  to quality trim the reads and aligned them to the closest strain Q154 . The raw coverage at this stage was 120×. Then, we used BayesHammer  to correct the Illumina reads followed by COPE  to merge overlapping reads (about 18%). The resulting merged and unmerged paired-end reads were assembled using Velvet-SC , SPAdes  and IDBA-UD , three assemblers optimized for single-cell sequence data with unequal coverage. Finally, we used GAM-NGS  to merge the contigs of the resulting assemblies. During the whole assembly process we used QUAST  to find the optimal parameters to obtain as few contigs as possible with the least number of misassemblies and InDels and the greatest N50 value. Afterwards, we used Mauve  to predict the order and orientation of the contigs corresponding to Q154.
A close inspection of the contig boundaries revealed many a lot of chimeric sequences, especially at nearly all of the possible IS1111 insertion sequence  sites. The reason for this frequent chimera formation can be explained by circular intermediates of IS1111 (although their presence in Nine Mile could not be detected by PCR, maybe caused by low expression level of these transposases) in combination with the whole genome amplification technique applied here [58, 59].
We used Cutadapt  to trim IS1111 sequences from the 5’ and 3’ end of the assembled contigs to avoid mis-scaffolding and artificial gap filling. Afterwards, we used Opera  and information from our extended IS1111 typing method (manuscript in preparation) to scaffold the contigs semi-automatically. Then we used GapFiller  to close or reduce gaps. All insertion sequences sites (including IS1111, IS30 and ISAs1) were verified again to avoid false positive insertions and to fill in missing sequences with Ns.
Gene calling and functional annotation was performed using the PEDANT system . Briefly, genes were called using Prodigal , Glimmer  and GeneMarkS ; all had been trained on the five RefSeq complete C. burnetii genomes. Consensus gene models were created by majority, domain or structural annotations in alternative start regions or preferring the Prodigal model. Structural RNAs were predicted using RNAmmer  (rRNAs), tRNAscan-SE  and similarity to Rfam . The known 23S rRNA intervening sequence (IVS)  and the two self-splicing group I introns  were annotated manually. Protein similarities and InterPro domain annotations were obtained from SIMAP  if possible or computed locally. Similarities to SCOP  and KEGG  were computed using BLAST . Signal peptides were predicted using SignalP , transmembrane proteins using TMHMM . Gene names and protein descriptions were annotated by a combination of a stringent similarity search against the UniProtKB/Swiss-Prot database  as well as using BLANNOTATOR  followed by manual curation. The genome and its functional annotation can be browsed at the PEDANT website .
Summary of genome: one chromosome and one plasmid
% of Total a
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, 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
A whole genome comparison with all five complete reference genomes (accessions: NC_009727, NC_010117, NC_002971, NC_011528, NC_011527) revealed strain Q154 as the most similar strain. In silico typing of strain Namibia showed the same adaA deletion variant 1 , but Multispacer Sequence Typing (MST) and Multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA) generated different profiles compared to Q154 (30 vs. 8 and D16 vs. D6 respectively) [46, 81].The COG distribution is quite similar, except fewer annotated proteins in Q154, likely because of older gene prediction algorithms. The tRNA composition is identical in both strains. At the nucleotide level 2,767 chromosomal SNPs and 77 plasmid-related SNPs were found (752 intergenic, 5 non-coding and 2,087 within coding regions). Further, there is a 6 kb region in the Namibia genome which is not present in the Q154/Q177 clade (Figure 2) but in the other complete reference strains. It contains the ankyrin repeat protein AnkI (CBNA_1063). Also, a 4.5 kb region present in Q154 (containing an acetyltransferase, CbuK_0095 and a bacterial regulatory protein, CbuK_0101) is absent in Namibia. Large structural variations were not detected.
We present the first whole genome sequence of Coxiella burnetii strain Namibia from Africa with its distinct genotype and unique genomic features and regions. We describe a combined set of laboratory methods and bioinformatics tools that resulted in a high quality whole genome sequence of this strain. The applied bioinformatics approach accounts for potential problems caused by the MDA/WGA method such as uneven sequence coverage and artificial products like chimeric reads. The sequencing and assembly pipeline presented here is suggested as a standard when sequencing of C. burnetii strains is done with or without the application of whole genome amplification methods. The incorporation of insertion sequence typing data can help to reduce the number of scaffolds down to a single whole genome sequence and avoids creating and sequencing an additional long distance mate-pair library usually needed to scaffold highly repetitive genomes.
To speed up the sequencing of new C. burnetii strains and to overcome the problems in generating high quality genomes, a joint research project with the Swedish Defence Institute (FOI) in Umeå was established: The Coxiella Genome Sequencing Consortium (CGSC) .
Whole genome amplification
Multiple displacement amplification
Buffalo Green Monkey.
This work was supported in part by the German Ministry of Education and Research (BMBF) under contract No. 01KI1001.
We would like to thank Heike Gehringer for critical reading and useful discussion of the manuscript.
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