Genomics and Ecophysiology of Heterotrophic Nitrogen-Fixing Bacteria Isolated from Estuarine Surface Water

The ability to reduce atmospheric nitrogen (N(2)) to ammonia, known as N(2) fixation, is a widely distributed trait among prokaryotes that accounts for an essential input of new N to a multitude of environments. Nitrogenase reductase gene (nifH) composition suggests that putative N(2)-fixing heterotrophic organisms are widespread in marine bacterioplankton, but their autecology and ecological significance are unknown. Here, we report genomic and ecophysiology data in relation to N(2) fixation by three environmentally relevant heterotrophic bacteria isolated from Baltic Sea surface water: Pseudomonas stutzeri strain BAL361 and Raoultella ornithinolytica strain BAL286, which are gammaproteobacteria, and Rhodopseudomonas palustris strain BAL398, an alphaproteobacterium. Genome sequencing revealed that all were metabolically versatile and that the gene clusters encoding the N(2) fixation complex varied in length and complexity between isolates. All three isolates could sustain growth by N(2) fixation in the absence of reactive N, and this fixation was stimulated by low concentrations of oxygen in all three organisms (≈4 to 40 µmol O(2) liter(−1)). P. stutzeri BAL361 did, however, fix N at up to 165 µmol O(2) liter(−1), presumably accommodated through aggregate formation. Glucose stimulated N(2) fixation in general, and reactive N repressed N(2) fixation, except that ammonium (NH(4)(+)) stimulated N(2) fixation in R. palustris BAL398, indicating the use of nitrogenase as an electron sink. The lack of correlations between nitrogenase reductase gene expression and ethylene (C(2)H(4)) production indicated tight posttranscriptional-level control. The N(2) fixation rates obtained suggested that, given the right conditions, these heterotrophic diazotrophs could contribute significantly to in situ rates.