NifH and NifD phylogenies: an evolutionary basis for understanding nitrogen fixation capabilities of methanotrophic bacteria

Abstract

The ability to utilize dinitrogen as a nitrogen source is an important phenotypic trait in most currently known methanotrophic bacteria (MB). This trait is especially important for acidophilic MB, which inhabit acidic oligotrophic environments, highly depleted in available nitrogen compounds. Phylogenetically, acidophilic MB are most closely related to heterotrophic dinitrogen-fixing bacteria of the genusBeijerinckia. To further explore the phylogenetic linkage between these metabolically different organisms, the sequences ofnifHandnifDgene fragments from acidophilic MB of the generaMethylocellaandMethylocapsa, and from representatives ofBeijerinckia, were determined. For reference,nifHandnifDsequences were also obtained from some type II MB of the alphaproteobacterialMethylosinus/Methylocystisgroup and from gammaproteobacterial type I MB. The trees constructed for the inferred amino acid sequences ofnifHandnifDwere highly congruent. The phylogenetic relationships among MB in the NifH and NifD trees also agreed well with the corresponding 16S rRNA-based phylogeny, except for two distinctive features. First, different methods used for phylogenetic analysis grouped the NifH and NifD sequences of strains of the gammaproteobacterial MBMethylococcus capsulatuswithin a clade mainly characterized byAlphaproteobacteria, including acidophilic MB and type II MB of theMethylosinus/Methylocystisgroup. From this and other genomic data fromMethylococcus capsulatusBath, it is proposed that an ancient event of lateral gene transfer was responsible for this aberrant branching. Second, the identity values of NifH and NifD sequences betweenMethylocapsa acidiphilaB2 and representatives ofBeijerinckiawere clearly higher (98·5 and 96·6 %, respectively) than would be expected from their 16S rRNA-based relationships. Possibly, these two bacteria originated from a common acidophilic dinitrogen-fixing ancestor, and were subject to similar evolutionary pressure with regard to nitrogen acquisition. This interpretation is corroborated by the observation that, in contrast to most other diazotrophs,M. acidiphilaB2 andBeijerinckiaspp. are capable of active growth on nitrogen-free media under fully aerobic conditions.