Genome of Acetobacter malorum DmelCS_005

Draft genome sequence for the fruit-fly gastrointestinal tract symbiont, Acetobacter malorum strain DmelCS_005

John M. Chaston, Peter D. Newell, Chun-nin (Adam) Wong, and Angela E. Douglas

 Department of Entomology, Cornell University, Ithaca, NY

Here we announce the sequencing of Acetobacter malorum isolated from the gastrointestinal tract of the fruit fly Drosophila melanogaster. We assembled the genome with Velvet and annotated the genome using the RAST server, attaining a draft genome sequence of 87 contigs with 3147 annotated features and a total of 3,516,804 bp.

Normal animal function is dependent upon resident microorganisms (‘microbiota’) that contribute to healthy host nutrition, immunity, and behavior. Over 100 traits relating to metabolic, nutritional, vascular, hepatic, respiratory, immunological, endocrine, and neural status differ between animal hosts that are raised in the presence or absence of colonizing bacteria (Bercik et al., 2011; Smith et al., 2007). In the fruit fly Drosophila melanogaster, colonizing bacteria affect diverse traits such as mating behavior (Sharon et al., 2010) and host nutritional indices (Ridley et al., 2012). We have previously identified Acetobacter malorum as one of six bacterial species that make up greater than 90% of the host gastrointestinal tract microbial community (Wong et al., 2011). A. malorum has also been identified in other surveys of D. melanogaster host-associated microbial communities (Chandler et al., 2011; Corby-Harris et al., 2007; Ryu et al., 2008). Here, we announce the sequencing of A. malorum DmelCS_005.

Acetobacter malorum was isolated from aseptically dissected guts of Drosophila melanogaster, and maintained at 30° C on M.R.S. medium (de Man et al., 1960).  Cultures were grown shaking at 200rpm to late-log phase and genomic DNA prepared with the Qiagen DNeasy Blood and Tissue Kit as per the manufacturer’s recommendations.  The Cornell Life Sciences Core Facility performed library preparation for Illumina sequencing.

We acquired 19,508,465, 100-bp paired-end read-pairs using a Illumina HiSeq 2000. 18,304,816 read-pairs passed quality filtering, providing ~1000X genome coverage, and we used a random subset of 17,500,000 quality filtered read pairs for the assembly. We assembled the genome using Velvet 1.2.03 (10) by randomly allocating each sequence to one of 7 subset files, each containing 2,500,000 read pairs (142X coverage per subset file). Sequences from each subset file were assembled into contigs using kmer lengths of 79, aided by predictions of the Velvet Advisor tool (Seeman) and tests with multiple kmer lengths. The output contig file from each subset was used as input in a second velvet run with a kmer length of 75 to create a final assembly that was 3,510,409 bp long and represented by 96 contigs, with a maximum contig size of 1,153,504-bp and N50 of 174,419-bp. Annotation and subsequent analyses were performed using the Rapid Annotation using Subsystem Technology (RAST) server (Aziz et al., 2008) to create an annotated genome sequence with 3,516,804 bp, 87 contigs, 3147 features, and an estimated 121 missing genes.

Based on genome sequence, we analyzed predicted functions of A. malorum in SEED (Overbeek et al., 2005). Consistent with expectations, there were complete or nearly complete predicted pathways for all of the essential amino acids genes. We found no predicted genes for bacterial motility or adhesion. We found few predicted iron acquisition and metabolism genes (including no siderophores) but 37 predicted TonB-dependent receptors. This is a similar number to the 35 TonB-dependent receptors present in Acetobacter tropicalis, another D. melanogaster gastrointestinal symbiont, and much greater than the number present in the D. melanogaster gastrointestinal tract symbiont Acetobacter pomorum (7 receptors) or in Escherichia coli (1 receptor) (data not shown). We did find predicted gene sequences involved in vitamin B12 synthesis, and the large number of TonB-dependent receptors may some connection with cobalamin, rather than iron-transport.

Acetobacter pasteurianus, the only Acetobacter species with a complete genome sequence, has 6 plasmids of varying length (Azuma et al., 2009). None of the assembled A. malorum contigs have full-length sequence similarity to any of the 6 A. pasteurianus plasmid sequences. However, two short contigs have similarity to genes that are likely plasmid genes and are present in one of the A. pasteurianus plasmids. Node 93 (4,519 bp long) contains regions with similarity to genes for RepA, 2 resolvases, and a plasmid partitioning protein. Node 37 (10,733 bp long) contains regions with similarity to traD and a conjugal transfer release protein A gene. These and other contigs may represent partial or complete plasmid sequences in A. malorum.

The sequence file is available as Download (see sidebar on left).  If you need the data in a different format, please contact us.

Acknowledgements

This work was supported by a NIH grant 1R01GM095372 to AED and Ruth L. Kirschstein NRSA postdoctoral fellowship to PDN (1F32GM099374-01). We thank Peter Schweitzer for assistance with filtering reads and Madeline Galac for assistance installing Velvet.

 

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