LB 7-71 - Identification of candidate enzymes for nitrite reduction in anaerobic ammonium oxidizing “Candidatus Kuenenia stuttgartiensis” strain CSTR1

Background/Question/Methods:

Anaerobic ammonium oxidizing (anammox) bacteria owe their position in the nitrogen cycle to their unique property to oxidize ammonium using nitrite as electron acceptor. In anammox strains belonging to the genus “Candidatus Kuenenia”, nitrite is first reduced to nitric oxide (NO) which is then used to oxidize ammonium to produce dinitrogen gas as the end product. Genome annotation revealed presence of a canonical heme protein known as nitrite reductase, NirS. However, the NirS is barely expressed at the transcriptional and proteomics levels. This put a question on how Candidatus Kuenenia reduces nitrite and which candidate enzymes perform this role. Here, we propose activity proteomics method and hypothesize that when we separate the whole proteome using fast protein liquid chromatography (FPLC) with size exclusion chromatography (SEC) or anion exchange chromatography (AEC), the active fraction that comes out should contain the catalytic enzyme for nitrite reduction. Therefore, we employed FPLC, in vitro enzymatic activity assay, Gas chromatography coupled to mass spectrometry (GC-MS), and quantitative proteomics to identify candidate enzymes for the nitrite reduction step in “Candidatus Kuenenia stuttgartiensis” strain CSTR1.

Results/Conclusions:

“Candidatus Kuenenia stuttgartiensis” strain CSTR1 cell extracts and fractions reduced nitrite to NO. FPLC with size exclusion chromatograph identified nitrite reduction activity in the fraction corresponding to a protein size of 150~200 kDa. FPLC with anion exchange chromatograph identified nitrite reduction activity in the fraction corresponding to a salt concentration of 300-500 mM NaCl. In both runs, active fractions were associated with high absorbance peaks of 408 nm which is characteristic of cytochrome proteins. Proteomics analyses of fractions in both runs revealed candidate enzymes that catalyze the reduction of nitrite in “Candidatus Kuenenia stuttgartiensis” strain CSTR1. In this activity assay, the reduction of nitrite to NO was not associated to NirS rather implicated hydroxylamine oxidase (HOX), NADP-dependent isopropanol dehydrogenase (ADH) and putative nitrite reductase electron transfer iron-sulfur -cluster subunit (4Fe-4S) to coincide with reduction of nitrite in CSTR1. The result also show that temperature and pH are important factors governing the activities and product formation of the nitrite reducing enzymes. Low pH (6.2) favors their activity more than neutral or basic pH while maximum activity was found at temperature within 60⁰C. This activity proteomics can expand our knowledge on respiratory processes in bacteria.