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Genome sequence of Algoriphagus PR1

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GENOME ANNOUNCEMENT

Complete Genome Sequence of Algoriphagus sp. PR1, Bacterial Prey of a Colonyforming Choanoflagellate

Rosanna A. Alegado1*, Steven Ferriera2,3, Chad Nusbaum3, Sarah K. Young3, Qian Zeng3, Alma Imamovic3, Stephen R. Fairclough1, Nicole King1

Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California 947201; J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 208502; Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts 021393

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Life Sciences Addition, #3200, Berkeley, CA 94720-3200 Phone: (510) 643-9417. Fax: (510) 643-6791. E-mail: rosie.alegado@berkeley.edu

Main text: 484 words (not including the title page, abstract, acknowledgements or references)

Keywords: Bacteroidetes, Algoriphagus, choanoflagellate, ATCC 50818, carbohydrateactive enzyme

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Abstract Bacteria are the primary food source of choanoflagellates, the closest known relatives of animals. The Gram-negative marine Bacteroidetes species Algoriphagus sp. PR1 was co-isolated with the choanoflagellate Salpingoeca rosetta from mud core samples near Hog Island, Virginia in May 2000 (14). Choanoflagellates are ideal organisms for studying the origin of animal multicellularity (13-15) and isolation of diverse bacterial food sources has facilitated the development of choanoflagellates as a new model system (16). Here we announce the complete genome sequence of Algoriphagus sp. PR1 and initial findings from its annotation.

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Bacteroidetes species comprise between 6-30% of total bacteria in the oceans (4, 11). Furthermore, they play an important role in the global carbon cycle because of their ability to degrade polysaccharides and other macromolecules (6, 8, 9, 25). Of the three clades that constitute the Bacteroidetes phylum (Cytophaga-Flavobacteria-Bacteroides), the Cytophaga clade, of which Algoriphagus is a member, has been least studied. The complete genome sequence of Algoriphagus sp. PR1 was determined using shotgun sequencing, 454 (19) and Illumina technologies (2). Initial assembly of a draft whole genome shotgun sequence into 12 contigs was generated at the JCVI based upon 50,413 Sanger sequencing reads from genomic libraries harboring 4-kb and 40-kb fragments. Resequencing of Algoriphagus sp. PR1 was performed at the Broad Institute and a 30x assembly containing a single gap was generated using the 454 Newbler assembler for 454 data (24), and the Velvet assembler (28) for Illumina data. The remaining gap is small and appears to be contained within a single gene. The Algoriphagus sp. PR1 genome was found to be a single circular 4.89 Mbp chromosome that is 38.69% GC rich, contains 3,954 predicted genes, and is similar in size to previously sequenced genomes from other marine Bacteroidetes (1, 21-23). Ab initio gene models were generated using GeneMark (3), Glimmer3 (7) and Metagene (20). Predicted genes were generated from BLAST hits to the UniRef90 database and a synteny-based approach was used to transfer ORFs from the draft PR1 genome. The final ORF set was derived by comparison of in silico ORFs, ORFs from BLAST hits and mapped ORFs with hits to Pfam (10), and the top BLAST hits against UniRef90. ORFs with overlap to non-coding RNA features were removed when appropriate. Discrepancies in the final ORFs were resolved manually. Non-coding features were identified with

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RNAmmer (17), tRNAScan (18) and RFAM (12). There are 39 transfer RNAs and 9 ribosomal RNA operons. The genome encodes genes required for a complete TCA cycle, and complete glycolysis and pentose phosphate pathways. Algoriphagus sp. PR1 forms pink-pigmented colonies and the genome encodes numerous carotenoid biosynthetic enzymes. Given the capacity of Bacteroidetes bacteria to degrade macromolecules, we catalogued the diversity of carbohydrate active enzymes in Algoriphagus sp. PR1. We found Algoriphagus sp. PR1 to have 62 glycoside hydrolases, 71 glycosyl transferases, 2 polysaccharide lyases and 10 carbohydrate esterases, constituting a high capacity for polysaccharide degradation. While the expansion of these groups of enzymes is a characteristic of the Bacteroidetes phylum (1, 7, 26, 27), Algoriphagus sp. PR1 possesses a repertoire more similar to gut commensal Bacteroidetes than marine Bacteroidetes, which may in part be related to its interactions with choanoflagellates. The sequencing and annotation of the Algoriphagus sp. PR1 genome provides a foundation for comparative studies of microbe-eukaryote interactions. Nucleotide sequence accession numbers. The JCVI genome sequence of Algoriphagus sp. PR1 is available in GenBank under access number AAXU01000000 and the Broad genome sequence is AAXU02000000.

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Acknowledgements The initial phase of sequencing, assembly, and annotation efforts were supported by a Gordon and Betty Moore Foundation Junior Investigator award (N.K.) and the Gordon and Betty Moore Foundation Marine Microbial Sequencing Project. Resequencing and genome finishing at the Broad Institute was supported by funding from NHGRI/NIH as part of the Origins of Multicellularity Project. Subsequent data analysis was conducted at UC Berkeley and supported by an NIH National Research Service Award and Fellowship grant to R.A.A. (5F32GM086054). N.K. is a Scholar in the Integrated Microbial Biodiversity Program of the Canadian Institute for Advanced Research.

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