Zooplankton in the Southern Ocean represent unique microhabitats for microbial communities that contribute significantly to polar biogeochemical cycles. In this study, we performed a polyphasic and genomic characterization of two Gram-negative, psychrotolerant bacterial strains, HL-MZ7T and HL-MZ19, isolated from Antarctic zooplankton. Phylogenetic and phylogenomic analyses clearly placed these isolates within the genus Pseudocolwellia, forming a sister clade to Pseudocolwellia agarivorans QM50T. The complete genomes of HL-MZ7T (4.5 Mbp) and HL-MZ19 (4.6 Mbp) exhibited DNA G + C contents of 36.0% and 35.9%, respectively. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values confirmed that both strains represent a novel genomic species, distinct from P. agarivorans QM50T (79.5% ANI and 24.0% dDDH). Comparative genomic analysis revealed a rich repertoire of genes associated with cold environmental adaptation and host-microbe interactions. Notably, we identified biosynthetic gene clusters of aryl polyenes and homoserine lactones, alongside genes involved in surface adhesion and nutrient acquisition, suggesting a specialized symbiotic potential within the zooplankton micro-niche. Phenotypically, the strains are characterized as strictly aerobic, with optimal growth at 15-20 °C and 3.0% (w/v) sea salts. These findings expand the genomic and ecological understanding of zooplankton-associated bacteria in polar marine ecosystems, and support the proposal of Pseudocolwellia antarctica sp. nov.
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