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

Abstract

The ability to reduce atmospheric nitrogen (N2) to ammonia, known as N2 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 N2-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 N2 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 N2 fixation complex varied in length and complexity between isolates. All three isolates could sustain growth by N2 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 O2 liter(-1)). P. stutzeri BAL361 did, however, fix N at up to 165 µmol O2 liter(-1), presumably accommodated through aggregate formation. Glucose stimulated N2 fixation in general, and reactive N repressed N2 fixation, except that ammonium (NH4 (+)) stimulated N2 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 (C2H4) production indicated tight posttranscriptional-level control. The N2 fixation rates obtained suggested that, given the right conditions, these heterotrophic diazotrophs could contribute significantly to in situ rates.

Importance: The biological process of importing atmospheric N2 is of paramount importance in terrestrial and aquatic ecosystems. In the oceans, a diverse array of prokaryotes seemingly carry the genetic capacity to perform this process, but lack of knowledge about their autecology and the factors that constrain their N2 fixation hamper an understanding of their ecological importance in marine waters. The present study documents a high variability of genomic and ecophysiological properties related to N2 fixation in three heterotrophic isolates obtained from estuarine surface waters and shows that these organisms fix N2 under a surprisingly broad range of conditions and at significant rates. The observed intricate regulation of N2 fixation for the isolates indicates that indigenous populations of heterotrophic diazotrophs have discrete strategies to cope with environmental controls of N2 fixation. Hence, community-level generalizations about the regulation of N2 fixation in marine heterotrophic bacterioplankton may be problematic.

FIG 1

Amino acid sequence-based, unrooted, neighbor-joining tree showing the affiliations of the isolates with the canonical nifH clusters (37). Color-coded lines indicate cluster affiliations as shown in the key. Pseudomonas stutzeri BAL361 represents the gammaproteobacterial part of cluster I, Rhodopseudomonas palustris BAL398 represents the alphaproteobacterial part of cluster I, and R. ornithinolytica BAL286 represents the gammaproteobacterial part of cluster I, as well as cluster II.

FIG 2

N₂ fixation gene clusters of Pseudomonas stutzeri BAL361 (A), Rhodopseudomonas palustris BAL398 (B), and Raoultella ornithinolytica BAL286 (C). Color-coded arrows indicate the locations of coding sequences (CDSs) and their orientations. The GC contents are depicted beneath the gene clusters (blue, GC content below cluster average; green, GC content above cluster average).

FIG 3

Growth of Pseudomonas stutzeri BAL361 (A), Rhodopseudomonas palustris BAL398 (B), and Raoultella ornithinolytica BAL286 (C) in carbonate-buffered microoxic diazotroph medium under standard conditions (20 mmol glucose liter⁻¹, 37 ± 4 µmol O₂ liter^{− 1}). Cell counts were obtained from each of the three cultures daily and are depicted from day 5 to 17 as means of values obtained within 48-h periods. Error bars represent standard deviations of the means. Pink bars indicate the 24-h period when cells were exposed to acetylene in the acetylene reduction assay.

FIG 4

Growth of Pseudomonas stutzeri BAL361 (A), Rhodopseudomonas palustris BAL398 (B), and Raoultella ornithinolytica BAL286 (C) in an oxygenated version of the growth medium (≈200 µmol O₂ liter⁻¹ at the time of inoculation). Cell counts were obtained from each of the three cultures daily and are depicted from day 5 to 17 as means of values obtained within 48-h periods. Error bars represent standard deviations of the means. (D) Cell-specific ethylene (C₂H₄) production rates as a function of the O₂ concentrations measured in the cultures at the time of C₂H₄ quantification. Horizontal and vertical error bars, where present, indicate the standard deviations of the mean O₂ concentration values measured at the time of C₂H₄ quantification and of the mean C₂H₄ production values, respectively.

FIG 5

Cell-specific ethylene (C₂H₄) production rates measured in triplicate serum vials as a function of eight glucose concentrations. Bulk C₂H₄ production did increase in R. ornithinolytica BAL286 cultures as a function of increasing glucose concentrations (not shown), but the higher number of cells meant a decrease in the cell-specific rates. Error bars indicate standard deviations of values from triplicate serum vials. Note the double logarithmic scale.

FIG 6

Cell-specific ethylene (C₂H₄) production rates measured in triplicate serum vials as a function of eight different concentrations of NO₃⁻ (A) and NH₄⁺ (B). (A) C₂H₄ production rates dropped 1 to 2 orders of magnitude for all three isolates when exposed to even low NO₃⁻ levels. (B) C₂H₄ production rates dropped for R. ornithinolytica BAL286 and P. stutzeri BAL361, while they increased significantly for R. palustris BAL398 compared to the rates for the other isolates when exposed to NH₄⁺ (P = 0.01). Error bars represent the standard deviations of values from triplicate serum vials, and lowercase letters indicate Tukey’s honestly significant difference (HSD) groupings. Note the logarithmic scale on the y axis.