Draft Genome Sequence of “Candidatus Spirobacillus cienkowskii,” a Pathogen of Freshwater Daphnia Species, Reconstructed from Hemolymph Metagenomic Reads

The species “Candidatus Spirobacillus cienkowskii” is a deep-branching uncultivated Deltaproteobacteria pathogen of freshwater daphniids, which are important members of aquatic food webs (1). The most distinctive phenotypic characteristic associated with its infection is the red color in the host hemolymph (2). We used high-throughput sequencing and computational binning approaches to assemble and reconstruct the pathogen genome, previously described only through sequences of 16S rRNA and DNA primase β-subunit (gyrB) genes (3).

Daphnia dentifera organisms infected with “Candidatus Spirobacillus cienkowskii” were originally collected from Dogwood Lake (38°32′37ʺN, 87°03′04ʺW; Greene-Sullivan State Forest, IN). Infections were propagated in D. dentifera L6D9 and the “standard” genotype collected from Dogwood Lake and a lake in Barry County, Michigan, respectively. The hemolymph from 43 infected hosts was collected and pooled for DNA extraction using the QIAamp DNA minikit (Qiagen, Germantown, MD) following manufacturer instructions. Metagenomic reads were generated with the Illumina MiSeq platform using paired-end 350-bp sequencing, and a total of 3,257,849 paired-end reads were obtained. Low-quality reads (<100 bp and a Phred score of <30) were filtered using Cutadapt v.1.18 (4), and the genome was assembled using IDBA-UD v.1.1.1 (5). A binning strategy was used to reconstruct the genome by taking into consideration the GC content and coverage of clustering contigs into individual genome populations through MaxBin v.2.0 (6) and manual curation with CheckM v.1.0.5 (7). To validate the genome reconstruction, BLASTN v.2.7.1+ (8) was used to compare nucleotide coding sequences (CDSs) to sequences of “Candidatus Spirobacillus cienkowskii” 16S rRNA (GenBank accession number EU220836) and gyrB (EU220837) genes deposited in GenBank.

Functional annotation was performed using PATRIC v.3.5.25 (9), Prokka v.1.12 (10), and KAAS v.1.0 (11). The “Candidatus Spirobacillus cienkowskii” genome was assembled into 126 contigs (2,739,001 bp with a GC content of 32.1%) with an N₅₀ value of 39,228 bp. Quality control of the genome assembly indicated a near-complete genome (91.2% completeness) without contamination (0%). Comparative analysis with available 16S rRNA and gyrB genes for “Candidatus Spirobacillus cienkowskii” showed 100% identities with our genome bin. A total of 2,553 CDSs, 37 tRNAs, and 1 complete rRNA operon (23S, 16S, and 5S rRNA) were detected in the genome. Of the total number of proteins, 37% had functional assignments (961 proteins), 15% had gene ontology assignments (402), and 46% had FIGfam assignments (1,176). Several genes associated with phytoene production (terpenoid backbone biosynthesis), the colorless precursor of all C₄₀ carotenoids (12), and other genes associated with carotenoid synthesis were detected (13). We identified genes associated with flagellar biosynthesis and assembly, which may be used for movement into the host hemolymph or for facilitating transmission to a new host.

Daphnia spp. are key members of lake food webs (14), and pathogen outbreaks reduce the host population growth rate and density and elevate the death rate (15). The “Candidatus Spirobacillus cienkowskii” genome will increase our knowledge of host-pathogen interactions. The annotated genome will help microbiologists identify conditions for isolating this ecologically important but as yet uncultivated pathogen.

We acknowledge the René Rachou Institute–Fiocruz Minas, Bioinformatics Platform, for computational logistics and support.

This work was supported by NSF DEB-1305836 to M.A.D.