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 genus Beijerinckia. To further explore the phylogenetic linkage between these metabolically different organisms, the sequences of nifH and nifD gene fragments from acidophilic MB of the genera Methylocella and Methylocapsa, and from representatives of Beijerinckia, were determined. For reference, nifH and nifD sequences were also obtained from some type II MB of the alphaproteobacterial Methylosinus/Methylocystis group and from gammaproteobacterial type I MB. The trees constructed for the inferred amino acid sequences of nifH and nifD were 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 MB Methylococcus capsulatus within a clade mainly characterized by Alphaproteobacteria, including acidophilic MB and type II MB of the Methylosinus/Methylocystis group. From this and other genomic data from Methylococcus capsulatus Bath, 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 between Methylocapsa acidiphila B2 and representatives of Beijerinckia were 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. acidiphila B2 and Beijerinckia spp. are capable of active growth on nitrogen-free media under fully aerobic conditions.
AumanA. J., SpeakeC. C., LidstromM. E.2001; nifH sequences and nitrogen fixation in type I and type II methanotrophs. Appl Environ Microbiol 67:4009–4016[CrossRef]
BoulyginaE. S., KuznetsovB. B., MarusinaA. I., TourovaT. P., KravchenkoI. K., BykovaS. A., KolganovaT. V., GalchenkoV. F.2002; A study of nucleotide sequences of nifH genes of some methanotrophic bacteria. Microbiology English translation of Mikrobiologiya 71:500–508
BürgmannH., WidmerF., von SiglerW., ZeyerJ.2004; New molecular screening tools for the analysis of free-living diazotrophs in soil. Appl Environ Microbiol 70:240–247[CrossRef]
DeanD. R., JacobsonM. R.1992; Biochemical genetics of nitrogenase. In Biological Nitrogen Fixation pp. 763–784Edited byStaceyG.BurrisR. H., EvansH. J. New York: Chapman & Hall;
DedyshS. N., LiesackW., KhmeleninaV. N., SuzinaN. E., TrotsenkoY. A., SemrauJ. D., BaresA. M., PanikovN. S., TiedjeJ. M.2000; Methylocella palustris gen. nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peat bogs, representing a novel subtype of serine-pathway methanotrophs. Int J Syst Evol Microbiol 50:955–969[CrossRef]
DedyshS. N., DerakshaniM., LiesackW.2001; Detection and enumeration of methanotrophs in acidic Sphagnum peat by 16S rRNA fluorescence in situ hybridisation, including the use of newly developed oligonucleotide probes forMethylocella palustris. Appl Environ Microbiol 67:4850–4857[CrossRef]
DedyshS. N., KhmeleninaV. N., SuzinaN. E., TrotsenkoY. A., SemrauJ. D., LiesackW., TiedjeJ. M.2002; Methylocapsa acidiphila gen. nov., sp. nov., a novel methane-oxidizing and dinitrogen-fixing acidophilic bacterium from Sphagnum bog. Int J Syst Evol Microbiol 52:251–261
DedyshS. N., DunfieldP. F., DerakshaniM., StubnerS., HeyerJ., LiesackW.2003; Differential detection of type II methanotrophic bacteria in acidic peatlands using newly developed 16S rRNA-targeted fluorescent oligonucleotide probes. FEMS Microbiol Ecol 43:299–308[CrossRef]
DunfieldP. F., KhmeleninaV. N., SuzinaN. E., TrotsenkoY. A., DedyshS. N.2003; Methylocella silvestris sp. nov., a novel methanotroph isolated from an acidic forest cambisol. Int J Syst Evol Microbiol 53:1231–1239[CrossRef]
FaniR., GalloR., LioP.2000; Molecular evolution of nitrogen fixation: the evolutionary history of the nifD, nifK, nifE, and nifN genes. J Mol Evol 51:1–11
HenneckeH., KaluzaK., ThönyB., FuhrmannM., LudwigW., StackebrandtE.1985; Concurrent evolution of nitrogenase genes and 16S rRNA in Rhizobium species and other nitrogen fixing bacteria. Arch Microbiol 142:342–348[CrossRef]
HeyerJ., GalchenkoV. F., DunfieldP. F.2002; Molecular phylogeny of type II methane-oxidizing bacteria isolated from various environments. Microbiology 148:2831–2846
KrumholzL. R., HollenbackJ. L., RoskesS. J., RingelbergD. B.1995; Methanogenesis and methanotrophy within a Sphagnum peatland. FEMS Microbiol Ecol 18:215–224[CrossRef]
LovellC. R., FriezM. J., LongshoreJ. W., BagwellC. E.2001; Recovery and phylogenetic analysis of nifH sequences from diazotrophic bacteria associated with dead aboveground biomass ofSpartina alterniflora. Appl Environ Microbiol 67:5308–5314[CrossRef]
MachadoI. M., YatesM. G., MachadoH. B., SouzaE. M., PedrosaF. O.1996; Cloning and sequencing of the nitrogenase structural genes nifHDK of Herbaspirillum seropedicae. Braz J Med Biol Res 29:1599–1602
NakamuraY., GojoboriT., IkemuraT.2000; Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucleic Acids Res 28:292[CrossRef]
ParkerM. A., LafayB., BurdonJ., van BerkumP.2002; Conflicting phylogeographic patterns in rRNA and nifD indicate regionally restricted gene transfer in Bradyrhizobium. Microbiology 148:2557–2565
RichardsonC. J., TiltonD. L., KadlecJ. A., ChamieJ. P. M., WentzW. A.1978; Nutrient dynamics of northern wetland ecosystems. In Freshwater Wetlands – Ecological Processes and Management Potential pp. 217–241Edited byGoodR. E.WhighamD. F., SimpsonR. L. New York: Academic Press;
RöschC., MergelA., BotheH.2002; Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil. Appl Environ Microbiol 68:3818–3829[CrossRef]
RuddJ. W. M., FurutaniA., FlettR. J., HamiltonR. D.1976; Factors controlling methane oxidation in shield lakes: the role of nitrogen fixation and oxygen concentration. Limnol Oceanogr 21:357–364[CrossRef]
SabraW., ZengA. P., LunsdorfH., DeckwerW. D.2000; Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase. Appl Environ Microbiol 66:4037–4044[CrossRef]
StrimmerK., von HaeselerA.1996; Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies. Mol Biol Evol 13:964–969[CrossRef]
SundhI., NilssonM., GranbergG., SvenssonB. H.1994; Depth distribution of microbial production and oxidation of methane in northern boreal peatlands. Microb Ecol 27:253–265
UedaT., SugaY., YahiroN., MatsuguchiT.1995b; Genetic diversity of N2 fixing bacteria associated with rice roots by molecular evolutionary analysis of a nifD library. Can J Microbiol 41:235–240[CrossRef]
WhittenburyR., PhillipsK. C., WilkinsonT. F.1970; Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol 61:205–218[CrossRef]
WhittenburyR.1981; The interrelationship of autotrophy and methylotrophy as seen in Methylococcus capsulatus (Bath). In Microbial growth on C1 compounds pp. 181–190Edited byDaltonH. London: Heyden;
WidmerF., ShafferB. T., PorteousL. A., SeidlerR. J.1999; Analysis of nifH gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon Cascade Mountain range. Appl Environ Microbiol 65:374–380
WoeseC. R., StackebrandtE., WeisburgW. G.8 other authors1984; The phylogeny of purple bacteria: the alpha subdivision. Syst Appl Microbiol 5:315–326[CrossRef]
YoungJ. P. W.1992; Phylogenetic classification of nitrogen-fixing organisms. In Biological Nitrogen Fixation pp. 43–86 Edited by StaceyG., BurrisR. H., EvansH. J. New York: Chapman & Hall;
YunA. C., SzalayA. A.1984; Structural genes of dinitrogenase and dinitrogenase reductase are transcribed from two separate promoters in the broad host range cowpea Rhizobium strain Irc78. Proc Natl Acad Sci U S A 81:7358–7362[CrossRef]
ZaniS., MellonM. T., CollierJ. L., ZehrJ. P.2000; Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR. Appl Environ Microbiol 66:3119–3124[CrossRef]
ZehrJ. P., McReynoldsL. A.1989; Use of degenerate oligonucleotides for amplification of nifH gene from the marine cyanobacteriumTrichodesmium thiebautii. Appl Environ Microbiol 55:2522–2526
ZehrJ. P., JenkinsB. D., ShortS. M., StewardG. F.2003; Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 5:539–554[CrossRef]
ZehrJ. P., MellonM. T., ZaniS.1998; New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nifH) genes. Appl Environ Microbiol 64:3444–3450