Extended Data Fig. 2: Organization and phylogeny of nitrogen fixation (nif) genes in ‘Candidatus Tectiglobus diatomicola’.

a, Organization of the nif regulon showing the phylogenetic affiliation and GC content of each gene. All nif genes are affiliated to gammaproteobacteria (yellow), whereas almost all other genes in and around the nif regulon are affiliated to alphaproteobacteria (blue). The presence of nifV indicates that unlike most nodulating rhizobia, ‘Ca. T. diatomicola’ has the capacity to synthesize homocitrate, a ligand of the FeMo cofactor⁶. Genes affiliated to gamma-and alphaproteobacteria were found together on individual PacBio reads (solid black lines). Note that there is no substantial difference in GC content between genes of gamma- and alphaproteobacterial origin. b, Maximum likelihood phylogenetic trees of NifHDKENBS sequences of the Pseudomonadota. Phylogeny is based on the alignment of full-length amino acid sequences retrieved from the GTDB. Cyanobacterial Nif sequences were used as outgroups. All ‘Ca. Tectiglobus diatomicola’/‘Ca. Tectiglobus profundi’ (pink line; 1) Nif protein sequences cluster with gammaproteobacterial sequences, while Nif sequences from other members of the Hyphomicrobiaceae family (purple lines; 2–9; Extended Data Fig. 3) cluster with alphaproteobacteria. These alphaproteobacterial Nif protein sequences form deeply branching sister clades to two of the major nodulating Rhizobiales nif clusters; i.e. Bradyrhizobium (I, solid line) and Allorhizobium-Mesorhizobium-Rhizobium-Sinorhizobium (II, dashed line). Note that some other Rhizobiales (turquoise lines; members of the Rhodobiaceae, Rhizobiaceae, Cohaesibacteraceae and BM303 families) Nif protein sequences also cluster with the gammaproteobacterial sequences indicating that they were also obtained via horizontal gene transfer from a (common) gammaproteobacterial donor. Tree scales indicate amino acid substitutions per site. Note that in panel A, individual nif genes are indicated by capital letters; fd, ferredoxin; hr, hemerythrin; hyp, hypothetical protein; prx, peroxiredoxin; rnd, ribonuclease D.