Table_5_Nitrogeniibacter aestuarii sp. nov., a Novel Nitrogen-Fixing Bacterium Affiliated to the Family Zoogloeaceae and Phylogeny of the Family Zoogloeaceae Revisited.DOCX

Members of the family Zoogloeaceae within the order Rhodocyclales are found to play vital roles in terrestrial and aquatic ecosystems by participating in biofloc formation in activated sludge, polycyclic aromatic hydrocarbon degradation, and nitrogen metabolism, such as denitrification and nitrogen fixation. Here, two bacterial strains designated H1-1-2AT and ZN11-R3-1 affiliated to the family Zoogloeaceae were isolated from coastal wetland habitats. The 16S rRNA gene sequences of the two strains were 100% identical and had maximum similarity with Nitrogeniibacter mangrovi M9-3-2T of 98.4% and ≤94.5% with other species. Phylogenetic analysis suggested that the two strains belonged to a single species and formed a novel monophyletic branch affiliated to the genus Nitrogeniibacter. The average nucleotide identity (ANI) value and digital DNA-DNA hybridization (dDDH) estimate between the two strains and N. mangrovi M9-3-2T were 78.5–78.7% and 21.4–21.6%, respectively, indicating that the two strains represent a novel species. The genomes of strain H1-1-2AT (complete genome) and ZN11-R3-1 (draft genome) were 4.7Mbp in length encoding ~4,360 functional genes. The DNA G+C content was 62.7%. Nitrogen fixation genes were found in the two strains, which were responsible for the growth on nitrogen-free medium, whereas denitrification genes found in N. mangrovi M9-3-2T were absent in the two strains. The respiratory quinone was ubiquinone-8. The major polar lipids consisted of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and aminophospholipid. The major fatty acids were summed feature 3 (C16:1ω7c and C16:1ω6c), C16:0, C12:0, and C10:0 3-OH. Based on genomic, phenotypic, and chemotaxonomic characterizations, strains H1-1-2AT and ZN11-R3-1 represent a novel species of the genus Nitrogeniibacter, for which the name Nitrogeniibacter aestuarii sp. nov. is proposed. The type strain is H1-1-2AT (=MCCC 1K04284T=KCTC 82672T), and additional strain is ZN11-R3-1 (=MCCC 1A17971=KCTC 82671). Additionally, phylogenomic analysis of the members of the family Zoogloeaceae including type strains and uncultivated bacteria was performed, using the Genome Taxonomic Database toolkit (GTDB-Tk). Combined with the 16S rRNA gene phylogeny, four novel genera, Parazoarcus gen. nov., Pseudazoarcus gen. nov., Pseudothauera gen. nov., and Cognatazoarcus gen. nov., were proposed. This study provided new insights to the taxonomy of the family Zoogloeaceae.

红环菌目（Rhodocyclales）下动胶菌科（Zoogloeaceae）的成员在陆地与水生生态系统中发挥关键作用，参与活性污泥生物絮凝体形成、多环芳烃降解及氮代谢过程（如反硝化作用与固氮作用）。本研究从滨海湿地生境中分离得到两株隶属于动胶菌科的细菌菌株，分别命名为H1-1-2AT与ZN11-R3-1。两株菌株的16S rRNA基因序列完全一致，与红树林固氮杆菌（Nitrogeniibacter mangrovi）M9-3-2T的最高相似性为98.4%，与其他物种的相似性则≤94.5%。系统发育分析显示，两株菌株属于同一物种，并形成了隶属于固氮杆菌属（Nitrogeniibacter）的全新单系分支。两株菌株与N. mangrovi M9-3-2T之间的平均核苷酸同源性（average nucleotide identity, ANI）值与数字DNA-DNA杂交（digital DNA-DNA hybridization, dDDH）估计值分别为78.5%~78.7%与21.4%~21.6%，表明两株菌株代表一个新物种。菌株H1-1-2AT（完成图基因组）与ZN11-R3-1（草图基因组）的基因组长度均为4.7 Mbp，编码约4360个功能基因，其DNA G+C含量为62.7%。两株菌株中携带固氮基因，使其可在无氮培养基上生长；而红树林固氮杆菌M9-3-2T所含的反硝化基因，在两株菌株中并未检出。该类菌株的呼吸醌为泛醌-8（ubiquinone-8），主要极性脂类包括磷脂酰乙醇胺、双磷脂酰甘油、磷脂酰甘油与氨基磷脂，主要脂肪酸为总和特征3（C16:1ω7c与C16:1ω6c）、C16:0、C12:0及C10:0 3-OH。基于基因组学、表型与化学分类学特征，菌株H1-1-2AT与ZN11-R3-1代表固氮杆菌属的一个新物种，故将其命名为滨海固氮杆菌（Nitrogeniibacter aestuarii）sp. nov.。模式菌株为H1-1-2AT（=MCCC 1K04284T=KCTC 82672T），另一株菌株为ZN11-R3-1（=MCCC 1A17971=KCTC 82671）。此外，本研究利用基因组分类学数据库工具包（Genome Taxonomic Database toolkit, GTDB-Tk），对包括模式菌株与未培养细菌在内的动胶菌科成员开展了系统基因组学分析。结合16S rRNA基因系统发育分析，本研究提议建立4个新属：副固氮弧菌属（Parazoarcus gen. nov.）、假固氮弧菌属（Pseudazoarcus gen. nov.）、假陶厄氏菌属（Pseudothauera gen. nov.）与近缘动胶菌属（Cognatazoarcus gen. nov.）。本研究为动胶菌科的分类学研究提供了新的见解。