Evidence for nitrogen fixation by "Dehalococcoides ethenogenes" strain 195

Abstract

Genome annotation of the chlorinated ethene-respiring "Dehalococcoides ethenogenes" strain 195 indicated the presence of a complete nitrogenase operon. Here, results from long-term growth experiments, gene expression, and (15)N(2)-isotope measurements confirm that strain 195 is capable of fixing atmospheric dinitrogen when a defined fixed-nitrogen source such as ammonium is unavailable.

FIG. 1.

(A) Dechlorination activity plotted as VC production and cell growth for strain 195 in medium with and without ammonium amendment. Cell growth data are averages of triplicate RT-qPCR analyses from each biological duplicate, and error bars represent 1 standard deviation. VC measurements are averages of biological duplicates, and error bars represent 5% uncertainty. The RT-qPCR method for 16S rRNA gene measurement has previously been described (11), and the chlorinated ethene concentrations were determined by gas chromatography-flame ionization detection (17). (B) Late-exponential-phase 16S rRNA, tceA, and nifD gene expression for cells growing with (day 10) and without (day 11) ammonium amendment. The results are averages of triplicate RT-qPCRs from each biological duplicate, and error bars represent 1 standard deviation.

FIG. 2.

Effects of ammonium addition on dechlorination activity (A) and cell growth (B) in ammonium-free cultures. The arrow indicates the time (day 15) when ammonium was added to the ammonium-free cultures.

FIG. 3.

nifD expression (A) and 16S rRNA gene and tceA expression (B) when cells were grown with or without ammonium amendment and the response to abrupt ammonium addition. The arrow indicates when the nitrogen-fixing cultures were amended with ammonium (day 15). No time zero point sample was collected for any transcription analysis, and nifD transcripts were unquantifiable at day 11.5 in all cultures. The analytical detection limit of nifD transcripts is 10⁻⁴ transcripts/gene as determined using the described RT-qPCR method and serial dilution of the quantified RNA standards.