Draft genome sequences of three rhodopsin possessing Croceitalea sp. strains, isolated from the sea surface microlayer in Japan

The genus Croceitalea belongs to the family Flavobacteriaceae in the phylum Bacteroidota. To date, four type species have been described in this genus, and members of this genus are characterized by Gram-negative, rod-shaped, yellow-orange colonies (1–3). Light-activated, ion-pumping rhodopsins are widely distributed among various bacteria and archaea which inhabit the photic zone of aquatic environments (4). Marine flavobacteria have been consistently found to possess rhodopsin genes, suggesting their ability to harness sunlight energy (5).

Strains used in this study were isolated from the sea surface microlayer (SML) of coastal Sagami Bay, Japan (35°09′04″N, 139°08′47″E). SML samples were collected by placing 47 mm polycarbonate membrane filters (Whatman, United Kingdom) onto the water surface for 10 s and spread onto 1/10-strength ZoBell agar plates (6–8). The plates were irradiated with UV-C (254 nm) at 595 μW/cm² for 60 s using a handy UV lamp (UVGL-25, Funakoshi, Japan) and incubated at 25°C for 2 weeks under white light condition. After incubation, the colonies were re-isolated onto 1/2 ZoBell agar plates, and three isolates (strains MTPC5, MTPC6, and MTPC9) were obtained. They were incubated in 10 mL of 1/10 ZoBell medium at 25°C for 1 week for DNA extraction. Genomic DNA samples were extracted using Wizard Genomic DNA purification kit (Promega, USA). Library preparation was performed with MGIEasy PCR-Free DNA Library PreSet and paired-end sequencing (2 × 150 bp) was performed with DNBSEQ-G400 Genetic Sequencer (MGI Tech, China) by Genome-Lead Ltd. (Kagawa, Japan). In total, 10,165,864 (MTPC5), 11,172,170 (MTPC6), and 12,989,703 (MTPC9) paired-end reads were obtained. The obtained reads were checked using FastQC v 0.12.0 (9) and trimmed using fastp v0.22.0 (10). The trimmed reads were assembled using Unicycler v0.4.8 (11). The assemblies were annotated using DDBJ Fast Annotation and Submission Tool (DFAST) v1.2.0 with the “Prodigal” and “tRNAscan-SE (Bacteria)” parameters (12). The qualities of the assemblies were checked using CheckM v1.2.2 (13). Average nucleotide identity (ANI) was calculated using the Genome-based distance matrix calculator (14). Digital DNA-DNA hybridization (dDDH) was calculated using the Genome-to-Genome Distance Calculator 3.0 (15).

The 16S rRNA gene sequence similarity values indicated that they belong to the genus Croceitalea, with the closest relatives being Croceitalea eckloniae DOKDO 025^T (MTPC5, 97.80%), Croceitalea marina H01-35^T (MTPC6, 96.84%), and C. marina H01-35^T (MTPC9, 96.84%), respectively. The ANI and dDDH values calculated using three genome sequences determined in this study consistently revealed 75.59%–77.35% ANI values and 17.4%–18.3% dDDH values against the public genomes of closely related strains: C. dokdonensis DOKDO 023^T (ASM130641v1), Croceitalea sp. F388 (ASM3184644v1), Croceitalea sp. P007 (ASM3184640v1), and Croceitalea sp. P059 (ASM3184636v1). MTPC6 and MTPC9 exhibited 100% similarity of both ANI and dDDH values, indicating they are likely the same species.

General genomic characteristics of these strains are listed in Table 1. Annotation results showed that these genomes share DNA repair and antioxidant genes, such as phr and sod which protect biomolecules from UV rays and oxidation (16, 17). Notably, each genome harbors a proteorhodopsin gene. This suggests that all three strains may have a photoheterotrophic lifestyle in the marine environment.

This work was supported by the Sasakawa Scientific Research Grant from The Japan Science Society (grant agreement number 02022-4096).