Genome of Lactobacillus fructivorans DmelCS_002

Draft genome sequence of Lactobacillus fructivorans DmelCS_002  isolated from the gut of Drosophila melanogaster

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

 Department of Entomology, Cornell University, Ithaca, NY

 

The bacterium Lactobacillus fructivorans DmelCS_002 is a prominent member of the gut microbiota of Drosophila melanogaster.  Here we report the draft genome sequence of Lactobacillus fructivorans DmelCS_002 .  We assembled the genome with Velvet and annotated the genome using the RAST server, attaining a genome sequence of 31 contigs with 1350 annotated features and a total of 1,333,245 bp.

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

Lactobacilli are the largest group in the Lactic Acid Bacteria (LAB), and can be found in many low oxygen, high nutrient environments (Ljungh and Wadstrom, 2009).  Lactobacilli are also frequent isolates from animal guts, including humans, mice, chickens and the fruit fly, Drosophila melanogaster (Kleerebezem and Vaughan, 2009; Walter, 2008; Wong et al., 2011).  Lactobacillus fructivorans is a heterofermentive LAB commonly associated with food spoilage (Ljungh and Wadstrom, 2009).  However, this species has also been isolated from fish and fruit fly guts (Picchietti et al., 2007; Ren et al., 2007; Wong et al., 2011).  In a comprehensive 454 sequencing of 16s rDNA amplicons from the fly gut, Wong et al. (2011) found L. fructivorans to be the dominant OTU present in early instar larvae and young adult flies.  The role of L. fructivorans association with flies has yet to be determined.   Here we report the genome sequence of Lactobacillus fructivorans DmelCS_002, which represents a useful resource for gaining insight into this understudied species from the fly gut microbiota. 

Lactobacillus fructivorans DmelCS_002 was isolated from aseptically dissected guts of Drosophila melanogaster, and maintained at 30° C on M.R.S. medium (de Man et al., 1960).  Bacteria were grown statically 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 Illumina library preparation and sequencing.

 We used 100 bp paired-end sequencing on an Illumina HiSeq 2000 and obtained sequence data for 17,267,008 read pairs. 16,324,275 read pairs passed quality filtering, providing us with >2,400x genome coverage. We assembled the genome using Velvet 1.2.03 (Zerbino and Birney, 2008) by randomly allocating 16,320,000 read pairs to one of 17 subset files each containing 960,000 read pairs (~144X coverage).  Sequences from each subset file were assembled into contigs using kmer lengths of 65-87, aided by predictions of the Velvet Advisor online tool (Zerbino and Birney, 2008). A representative contig file from each subset was used as input in a second velvet run with a kmer length of 75-85 to create a final assembly that was 1,333,965 bp in length, represented by 38 contigs, with a maximum contig size of 357,852 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 1,333,245 bp, 31 contigs, and 1350 features.

Preliminary comparisons between our genome and that of the only other genome-sequenced representative of the species, L. fructivorans KCTC 3243, indicate a high degree of similarity.  We ran a blastP search of the KCTC 3243 genome with each of 1,281 predicted proteins from the DMC001 sequence, and found 47 putative “unique” sequences (E value of best match >0.01).  Of these, 12 had no match in the NCBI non-redundant protein database (NRDB), suggesting that they are spurious predictions.  The remaining 35 produced NCBI NRDB hits with E values ≤ 3x10-5, and include a number of interesting predicted functions: five transporters, two transcriptional regulators, two transposases, two phage or prophage proteins, a large LPXTG-motif surface protein, and a CRISPR region with five CAS proteins.  Top BLAST hits from the CAS proteins suggest that this locus is homologus to CRISPR loci in a number of other lactobacilli, and belongs to the Csn1-type CRISPR family (Horvath et al., 2009).  The large LPXTG-motif surface protein is a serine-rich repeat family protein, 3,377 amino acids in length.  It bears structural similarity to biofilm adhesins in streptococci and other Gram-positive relatives (Nobbs et al., 2009; Zhou and Wu, 2009), but appears to have no homolog in the Lactobacilli.

 

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

 

References

Aziz, R.K., Bartels, D., Best, A.A., DeJongh, M., Disz, T., Edwards, R.A., Formsma, K., Gerdes, S., Glass, E.M., Kubal, M., et al. (2008). The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9, 75.

de Man, J.D., Rogosa, M., and Sharpe, M.E. (1960). A medium for the cultivation of Lactobacilli. Journal of Applied Bacteriology 23, 130-135.

Horvath, P., Coute-Monvoisin, A.C., Romero, D.A., Boyaval, P., Fremaux, C., and Barrangou, R. (2009). Comparative analysis of CRISPR loci in lactic acid bacteria genomes. International journal of food microbiology 131, 62-70.

Kleerebezem, M., and Vaughan, E.E. (2009). Probiotic and gut lactobacilli and bifidobacteria: molecular approaches to study diversity and activity. Annual review of microbiology 63, 269-290.

Ljungh, A., and Wadstrom, T., eds. (2009). Lactobacillus Molecular Biology: from Genomics to Probiotics (Norfolk, UK: Caister Academic).

Nobbs, A.H., Lamont, R.J., and Jenkinson, H.F. (2009). Streptococcus adherence and colonization. Microbiol Mol Biol Rev 73, 407-450, Table of Contents.

Picchietti, S., Mazzini, M., Taddei, A.R., Renna, R., Fausto, A.M., Mulero, V., Carnevali, O., Cresci, A., and Abelli, L. (2007). Effects of administration of probiotic strains on GALT of larval gilthead seabream: Immunohistochemical and ultrastructural studies. Fish & shellfish immunology 22, 57-67.

Ren, C., Webster, P., Finkel, S.E., and Tower, J. (2007). Increased internal and external bacterial load during Drosophila aging without life-span trade-off. Cell Metab 6, 144-152.

Walter, J. (2008). Ecological role of lactobacilli in the gastrointestinal tract: implications for fundamental and biomedical research. Appl Environ Microbiol 74, 4985-4996.

Wong, C.N., Ng, P., and Douglas, A.E. (2011). Low-diversity bacterial community in the gut of the fruitfly Drosophila melanogaster. Environ Microbiol 13, 1889-1900.

Zerbino, D.R., and Birney, E. (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome research 18, 821-829.

Zhou, M., and Wu, H. (2009). Glycosylation and biogenesis of a family of serine-rich bacterial adhesins. Microbiology 155, 317-327.