Draft Genome Sequences of Pseudomonas moraviensis UCD-KL30, Vibrio ostreicida UCD-KL16, Colwellia sp. Strain UCD-KL20, Shewanella sp. Strain UCD-KL12, and Shewanella sp. Strain UCD-KL21, Isolated from Seagrass

ABSTRACT Here, we present the draft genome sequences for five bacterial strains. These strains were all isolated from seagrass (Zostera marina) collected from Bodega Bay, CA, as a part of an undergraduate research project focused on seagrass-associated microbes.

A s part of the seagrass microbiome project (https://seagrassmicrobiome.org/), bacterial isolates were cultured from seagrass (Zostera marina) and surrounding sediment. In order to determine which isolates would undergo genome sequencing, we used the general protocol for identifying isolates used by Dunitz et al. (1). Pseudomonas moraviensis UCD-KL30 was isolated from seagrass leaf scrapings placed in phosphatebuffered saline (PBS), which was plated onto nitrogen-free agar (15 g/liter agar, 1 g/liter CaCO 3 , 1 g/liter K 2 HPO 4 , 0.2 g/liter MgSO 4 , 0.2 g/liter NaCl, 0.1 g/liter FeSO 4 , 5 g/liter Na 2 MoO 4 , 50 ml 1:50 [wt/vol] glucose) and left them at 25°C for 2 weeks. The remaining isolates were cultured on Difco marine broth agar plates. Isolate Shewanella sp. UCD-KL12 was selected from a PBS rinse of a scraped seagrass leaf, which was cultured at 4°C for 3 weeks. Isolates Colwellia sp. UCD-KL20 and Shewanella sp. UCD-KL21 were obtained from a single dilution of seagrass sediment that was cultured for a week at 25°C. Vibrio ostreicida was selected from a PBS rinse of a seagrass leaf cultured for 2 days at 25°C. Kept at their respective temperatures, Difco marine broth was used to create liquid overnight cultures for all five isolates.
Following the genomic DNA extraction, to complete the whole-genome sequencing, paired-end libraries were created using a Nextera XT library preparation kit (Illumina). This size-selected library (600 to 900 bp) was sequenced on a paired-end 300-bp run of an Illumina MiSeq. Quality trimming error correction and assembly were performed using the A5-miseq assembly pipeline (2, 3). Genome completeness was estimated using PhyloSift, which revealed that each assembly contained single copies of 37 conserved single-copy marker genes (2). For all genomes, annotation was completed using RAST (4). The results for each assembly and annotation can be found in Table 1.
Full-length 16S rRNA gene sequences were retrieved from the RAST annotation and then used in RDP (5) to create alignments for each isolate and their close relatives. In order to determine the taxonomy, RDP was also used to obtain 16S rRNA gene sequences for close relatives and used in the creation of the phylogenetic trees (5). All trees were inferred using FastTree (6), visualized in Dendroscope (7), and can be found For the Colwellia and Shewanella isolates, the alignments were used to infer a maximum likelihood 16S rRNA tree using data from close relatives. For all three strains, the species-level taxonomy was ambiguous, and we did not assign species names to these isolates. Similar analysis for UCD-KL16 resulted in a well-supported clade that contained multiple other V. ostreicidia strains that were not found anywhere else in the tree. For UCD-KL30, a 16S rRNA gene phylogenetic tree proved to be uninformative. Therefore, we created a concatenated 37-marker tree of this strain and other sequenced relatives. The resulting tree reveals an error in taxonomy, a strain of Pseudomonas koreensis within a clearly delineated clade of P. moraviensis. We confirmed this misidentification using an average nucleotide identity (ANI) comparison of these strains (8).
Accession number(s). These genome sequences are available under the accession numbers provided in Table 1.

ACKNOWLEDGMENTS
Sequencing was performed at the DNA Technologies Core, University of California, Davis, Davis, CA. This work was funded by a grant from the Gordon and Betty Moore Foundation (GBMF333), "Investigating the co-evolutionary relationships between seagrasses and their microbial symbionts."