Polyphasic taxonomic characterization of two novel species from agricultural water, Chitinimonas fontana sp. nov., and Roseateles fontanus sp. nov.

Two Gram-stain-negative, rod-shaped, non-motile bacterial strains, designated BJYL2 T and BJYL3 T , were isolated from an agricultural pond and characterized using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BJYL2 T was affiliated with the genus Chitinimonas , whereas strain BJYL3 T belonged to the genus Roseateles . Strain BJYL2 T showed the highest 16 S rRNA gene sequence similarity to Chitinimonas prasina LY03 T (97.73%), and strain BJYL3 T showed the highest similarity to Roseateles saccharophilus DSM 654 T (97.72%). Whole-genome comparisons based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) showed values below the accepted species delineation thresholds, supporting the proposal that the two strains represent novel species within their respective genera. Both strains grew optimally at 30 °C and were oxidase-positive and catalase-negative. The predominant respiratory quinone was Q-8, and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The genomic DNA G + C contents were 61.29 mol% for BJYL2 T and 69.11 mol% for BJYL3 T . Genome analysis revealed multiple carbohydrate-active enzyme (CAZyme) families potentially involved in chitin utilization. These genomic features suggest that both strains have substantial genetic potential to utilize chitin-derived substrates. This trait may be ecologically relevant given the abundance of chitin sources in agricultural freshwater environments. Strain BJYL2 T contained three GH18 genes and one GH3 gene, whereas strain BJYL3 T possessed four GH18 genes, one GH19 gene, and two GH3 genes. Both genomes also contained several CE4 and GH23 family genes, although the specific roles of these genes in chitin utilization require further functional validation. Domain architecture analysis further indicated that several chitinase-related proteins contain carbohydrate-binding modules and secretion-associated signal sequences, suggesting the potential for extracellular chitin degradation. Quantitative assays further confirmed detectable chitinase activity in both strains, with BJYL2 T yielding 661.3 ± 44.2 and 561.1 ± 13.6 µg mL −1 GlcNAc after 3 and 5 days, respectively, and BJYL3 T yielding 506.3 ± 45.7 and 66.3 ± 2.1 µg mL −1 at the corresponding time points, compared with 39.2 ± 0.4 and 41.4 ± 0.8 µg mL −1 for the negative control. Based on the phenotypic, chemotaxonomic, phylogenetic, and genomic evidence, strains BJYL2 T and BJYL3 T represent novel species within the genera Chitinimonas and Roseateles , respectively.