Genome sequence of Candidatus Arsenophonus lipopteni, the exclusive symbiont of a blood sucking fly Lipoptena cervi (Diptera: Hippoboscidae)

Classification and features

Ca. Arsenophonus lipopteni has an obligate association with its host, L. cervi , and is therefore uncultivable. In order to localize the bacteria within the host, Fluorescent In Situ Hybridization and Transmission Electron Microscopy was performed on dissected gut tissue as described in detail in [7]. For FISH, the tissue was fixed and hybridized in tubes with eubacterial (EUB338, Flc-GCTGCCTCCCGTAGGA; [10]) and Ca. Arsenophonus lipopteni specific probes (ArL, Cy3-CTGACTAACGCTTGCACC; this study). The later was designed in a variable region of 16S rRNA gene taking the target sequence accessibility into account [11].

The distribution of Ca. Arsenophonus lipopteni (Fig. 1) in the host body closely resembles that of Ca. Arsenophonus melophagi and Wigglesworthia glossinidia ^T , the obligate symbionts of the blood sucking flies Melophagus ovinus and Glossina sp., respectively [7]. Highly pleomorphic cells of the Gram negative non-sporulating bacteria from the family Enterobacteriales are primarily found in the modified part of the gut wall (bacteriome) formed by the specialized enlarged epithelial cells (bacteriocytes, Fig. 1c, 1d). Additional key features of Ca. Arsenophonus lipopteni are provided as a standardized summary in Table 1.

MIGS ID	Property	Term	Evidence codea
	Classification	Domain Bacteria	TAS	[33]
		Phylum Proteobacteria	TAS	[34]
		Class Gammaproteobacteria	TAS	[35]
		Order “Enterobacteriales”	TAS	[36]
		Family Enterobacteriaceae	TAS	[37]
		Genus Arsenophonus	TAS	[38]
		Species Ca. Arsenophonus lipopteni	IDA
		Strain: CB	IDA
	Gram stain	Negative	TAS	[38]
	Cell shape	Pleomorphic	NAS
	Motility	Non-motile	TAS	[38]
	Sporulation	Non-sporulating	TAS	[38]
	Temperature range	Not determined	IDA
	Optimum temperature	Not determined	IDA
	pH range; Optimum	Not determined	IDA
	Carbon source	Not determined	IDA
MIGS-6	Habitat	Insect host; bacteriome of L. cervi	IDA
MIGS-6.3	Salinity	Not determined	IDA
MIGS-22	Oxygen requirement	Not determined	IDA
MIGS-15	Biotic relationship	Symbiotic	IDA
MIGS-14	Pathogenicity	Non-pathogen	NAS
MIGS-4	Geographic location	Ceske Budejovice, Czech Republic	IDA
MIGS-5	Sample collection date	June 2010	IDA
MIGS-4.1	Longitude	14.43	IDA
MIGS-4.2	Latitude	48.97	IDA
MIGS-4.4	Altitude	399 m	IDA

^aEvidence codes, IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature), NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [39]

Apart from the functional characterization, the genome sequence of Ca. Arsenophonus lipopteni was also utilized to assess the relationship of this bacterium to other Arsenophonus symbionts. Since the sequence compositional shift compromises phylogenetic usage of 16S rDNA, leading to topological artifacts with long branched symbiotic taxa clustering together [12], we carried out a phylogenetic analysis of a multi-gene matrix and used advanced Bayesian approaches. The matrix was generated for all available Arsenophonus genomes (incl. Ca. Riesia pediculicola ), five other symbionts, eight non-symbiotic members of Enterobacteriaceae , and two outgroups. A set of 70 orthologous genes was determined as an intersection of COGs shared by these bacteria (generated using the MicrobesOnline database; [13]) with “SICO” gene list [14]. The genes were retrieved from the finished assembly using Blastp searches [15] and processed as described previously [7]. The resulting matrix contained 22618 unequivocally aligned positions. Phylobayes [16], a tool specifically developed to overcome the difficulty with heterogeneous composition of sequences, was used for the tree reconstruction. The analysis was run in 2 chains under the GTR + CAT model with amino acids recoded according to the Dayhoff6 option. When the convergence was not reached after 20,000 cycles, the program was stopped and majority rule consensus was calculated after discarding 4,000 cycles burn-in.

The results confirm Ca. Arsenophonus lipopteni membership in the genus Arsenophonus . All Arsenophonus species (including Ca. Rieisa pediculicola) formed a well-supported monophyletic branch clustering as a sister group to Providencia (Fig. 2). Despite the length of branches for the obligate symbionts with highly modified genomes, this arrangement was assigned a high posterior probability. Although the six included Arsenophonus lineages certainly do not form a monophyletic group within the known Arsenophonus spectrum [17], the results indicate that Ca. Arsenophonus lipopteni evolved independently from Ca. Arsenophonus melophagi housed in related Hippoboscidae host [7].

Genome project history

Growth conditions and genomic DNA preparation

Since the bacterium is uncultivable, the host tissue was used for DNA extraction. The gut tissue containing the symbiotic organs were dissected from 6 flies in 1× phosphate buffered saline, homogenized with a sterile mortar and pestle and extracted using QiaAmp DNA Micro Kit (QIAGEN, United Kingdom). The DNA quality was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies).

Genome sequencing and assembly

The paired end 100 bp long reads were generated on one lane of Illumina HiSeq2000 run at Yale Center for Genome Analysis. A5 assembly pipeline with the default settings was used to assemble the reads [18]. Of the 109,640 resulting contigs, the longest contig (836,730 bp) with 40× fold coverage formed a circular molecule with 99 bp overlap at the ends. This contig corresponds to the Ca. Arsenophonus lipopteni genome. Pilon v1.12 [19] was used to check assembly quality and to improve base calls and small indels.

Genome annotation

The finished genome was annotated using a combination of following tools: RAST [20], PGAP, and Prokka v1.10 [21]. The annotation was then manually curated and checked for the presence of gene remnants. The final annotation is available in GenBank (CP013920). Metabolic pathways were reconstructed in the RAST server [20] and gene absence was verified using BlastP searches. Proteins were assigned to the clusters of orthologous groups using COGnitor [22], and the presence of signal peptides was detected using SignalP [23]. Pfam domains were predicted using HMMER [24] against the Pfam-A database [25]. Transmembrane predictions were done using TMHMM Server v. 2.0. The search for CRISPR repeats was performed in Geneious [26].