Extensive genomic study characterizing three populations and revealing sp. nov. and sp. nov.

Bacteria within the family show promising potential for applications in various fields, garnering significant research attention. Three Gram stain-negative bacteria, strains CPCC 101601, CPCC 101403, and CPCC 100767, were isolated from diverse environments: freshwater, rhizosphere soil of , and the phycosphere, respectively. Analysis of their 16S rRNA gene sequences, compared with those in the GenBank database, indicated that they belong to the family , with nucleotide similarities of 92.5%-99.9% to all of the members with valid taxonomic names. Phylogenetic studies based on 16S rRNA gene and whole-genome sequences identified CPCC 101601 as a member of the genus , CPCC 101403 belonging to the genus , and CPCC 100767 as part of the genus . Notably, genomic analysis using average nucleotide identity (ANI; <95%) and digital DNA-DNA hybridization (dDDH; <70%) with their closely related strains suggested that CPCC 101601 and CPCC 101403 represent new species within their respective genera. Conversely, CPCC 100767 exhibited high ANI (98.5%) and dDDH (87.4%) values with con5, indicating it belongs to this already recognized species. The in-depth genomic analysis revealed that strains CPCC 101601, CPCC 101403, and CPCC 100767 harbor key genes related to the pathways for denitrifying, MA utilization, and polyhydroxyalkanoate biosynthesis. Moreover, genotyping and phenotyping analysis confirmed that strain CPCC 100767 has the ability to convert atmospheric nitrogen into ammonia and produce 5-aminolevulinic acid, whereas CPCC 101601 can only perform the former bioprocess.IMPORTANCEBased on polyphasic taxonomic study, two new species, and affiliated with the family were identified. This expands our understanding of the family and provides new microbial materials for further studies. Modern genomic techniques such as average nucleotide identity and digital DNA-DNA hybridization were utilized to determine species affiliations. These methods offer more precise results than traditional classification mainly based on 16S rRNA gene analysis. Beyond classification of these strains, the research delved into their genomes and discovered key genes related to denitrification, MA utilization, and polyhydroxyalkanoate biosynthesis. The identification of these genes provides a molecular basis for understanding the environmental roles of these strains. Particularly, strain CPCC 100767 demonstrated the ability to convert atmospheric nitrogen into ammonia and produce 5-aminolevulinic acid. These bioprocess capabilities are of significant practical value, such as in agricultural production for use as biofertilizers or biostimulants.