The bacterial group of the phylum Bacteroidota greatly contributes to the global carbon cycle in marine ecosystems through its specialized ability to degrade marine polysaccharides. In this study, it is proposed that two novel facultative anaerobic strains, DS1-an-13321T and DS1-an-2312T, which were isolated from a sea squirt, represent a novel genus, Halosquirtibacter, with two novel species in the family Prolixibacteraceae. The 16S rRNA sequence similarities of these two strains were 91.26% and 91.37%, respectively, against Puteibacter caeruleilacunae JC036T, which is the closest recognized neighbor. The complete genomes of strains DS1-an-13321T and DS1-an-2312T each consisted of a single circular chromosome with a size of 4.47 and 5.19 Mb, respectively. The average amino acid identity and the percentage of conserved proteins against the type species of the genera in the family Prolixibacteraceae ranged from 48.33 to 52.35% and 28.34–37.37%, respectively, which are lower than the threshold for genus demarcation. Strains DS1-an-13321T and DS1-an-2312T could grow on galactose, glucose, maltose, lactose, sucrose, laminarin, and starch, and only DS1-an-2312T could grow on xylose and xylan under fermentation conditions. These strains produced acetic acid and propionic acid as the major fermentation products. Genome mining of the genomes of the two strains revealed 27 and 34 polysaccharide utilization loci, which included 155 and 249 carbohydrate-active enzymes (CAZymes), covering 57 and 65 CAZymes families, respectively. The laminarin-degrading enzymes in both strains were cell-associated, and showed exo-hydrolytic activity releasing glucose as a major product. The xylan-degrading enzymes of strain DS1-an-2312T was also cell-associated, and had endo-hydrolytic activities, releasing xylotriose and xylotetraose as major products. The evidence from phenotypic, biochemical, chemotaxonomic, and genomic characteristics supported the proposal of a novel genus with two novel species in the family Prolixibacteraceae, for which the names Halosquirtibacter laminarini gen. nov., sp. nov. and Halosquirtibacter xylanolyticus sp. nov. are proposed. The type strain of Halosquirtibacter laminarini is DS1-an-13321T (= KCTC 25031T = DSM 115329T) and the type strain of Halosquirtibacter xylanolyticus is DS1-an-2312T (= KCTC 25032T = DSM 115328T).
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