Draft Genome Sequence of a Novel Species of Halococcus (Strain IIIV-5B), an Endophytic Archaeon Isolated from the Leaf Tissue of Avicennia germinans

Strain IIIV-5B was isolated from the leaf tissue of the black mangrove, Avicennia germinans. This microorganism belongs to the genus Halococcus. Here, we present the draft genome sequence of strain IIIV-5B, a novel species of this genus. The 3,869,808-bp genome has a G+C content of 63.9% and around 3,812 coding sequences.

M angroves are woody plants often located in the intertidal areas of tropical and subtropical regions (1) that provide a suitable habitat for multiple species and contribute to the stabilization of coastlines by preventing erosion and protecting the shore from tidal waves (2)(3)(4)(5)(6)(7)(8). A novel strain (IIIV-5B) belonging to the genus Halococcus, first reported by Schoop in 1935 (9), was isolated and characterized during a prokaryotic biodiversity survey of halophilic and halotolerant endophytes in leaves of Avicennia germinans at the solar salterns in Cabo Rojo, Puerto Rico. Here, we present the draft genome sequence of this putative novel species, as reports of endophytic haloarchaea are scarce.
Strain IIIV-5B was isolated from surface-sterilized leaf tissue in Sehgal-Gibbons medium (10) containing 15% NaCl (wt/vol). The leaf tissue was placed onto the surface of solid medium and incubated at 30°C. Growing colonies were selected and purified by plate streaking until pure cultures were obtained. Genomic DNA extraction was performed using the Promega Wizard genomic DNA purification kit and sequenced at MicrobesNG in Birmingham, United Kingdom. Genomic DNA libraries were generated using a Nextera XT library prep kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions, with the exception of the use of 2 ng of DNA instead of 1 ng as input and an increase of the PCR elongation time to 1 min. Pooled libraries were quantified using the Kapa Biosystems library quantification kit for Illumina. Libraries were sequenced using an Illumina HiSeq instrument (250-bp paired-end protocol). Adapters were trimmed with Trimmomatic 0.30 (11), de novo assembly was performed using SPAdes version 3.7 (12), and contigs were annotated with Prokka 1.11 (13). Genome sequences were then analyzed using the Rapid Annotations using Subsystems Technology (RAST) server (14)(15)(16). A draft genome sequence of 3,869,808 bp was assembled using 74 contigs (Table 1). Features in this genome include a GϩC content of 63.96%, 2 CRISPR repeats, and around 3,812 coding sequences, 3 of which are suggested to encode rRNAs and 49 of which are suggested to encode tRNAs. Similar values were obtained with the NCBI Prokaryotic Genome Annotation Pipeline (3,877,752 bp, 63.9% GϩC content, and 3,812 coding sequences).
Sequences corresponding to the 16S rRNA and rpoB= genes were retrieved from the IIIV-5B annotated genome to perform taxonomical characterization. To determine the phylogenetic distance from other Halococcus spp., the 16S rRNA gene was obtained from the RAST server and uploaded to EZ-Taxon (17), where taxonomically close relatives were retrieved. 16S rRNA gene sequences were downloaded, aligned using ClustalW, and edited in Molecular Evolutionary Genetics Analysis X (MEGA X) software (18). The neighbor-joining tree method (19) was used to determine phylogenetic  distances of 16S rRNA and rpoB= gene sequences between IIIV-5B and closely related strains ( Fig. 1A and B). Default parameters were used for all software. The 16S rRNA gene of IIIV-5B was found to be 99.5% identical to that of Halococcus salsus ZJ1. Phylogenetic analysis of the rpoB= gene suggests that strain IIIV-5B might represent a new species of Halococcus. Data availability. The draft genome sequence of strain IIIV-5B has been deposited in GenBank under the accession number QZWE00000000. The raw data have been deposited in the SRA under the accession number PRJNA490534.

ACKNOWLEDGMENTS
We thank members of the Extremophile Laboratory at the UPRM for their help, especially Javier Rentas-Colón, Jessalyn Pla-Tenorio, and Carlos Vega-Chacón. Special thanks go to Ricardo Couto-Rodríguez, Carlos Rodríguez, and Carlos Santos for their contributions to and comments on this study.
This study was supported by the Howard Hughes Medical Institute (HHMI) project "Enhancing Advanced Educational Opportunities in STEM Fields for Minority Students at UPRM," grant number 52007566.