An indispensable protein catalyzing nitrite reduction in "Candidatus Kuenenia stuttgartiensis" strain CSTR1: activity and physiological properties

The physiological enzyme that reduce nitrite in anaerobic ammonium oxidizing (anammox) bacteria belonging to "Candidatus Kuenenia stuttgartiensis" is a mystery. The ongoing curiosity in identifying this enzyme has led to the proposal of myriads of proteins as potential candidates. So far, most of the studies on nitrite reductase in "Ca. Kuenenia" were centred only on purified proteins which mostly resulted in inconclusive evidence. However, crude cell extract can be a platform for identification of major protein candidates and their physiological properties. Here, we performed a comprehensive screening of nitrite-reducing activity in the proteome of "Ca. Kuenenia stuttgartiensis" strain CSTR1 which was dominant in a continuous stirred tank reactor (CSTR) containing planktonic cells. We hypothesized that a fast and reliable methods capable of separating the proteins in crude cell extract would enable a broader evaluation of the protein activities. In this context, fast protein liquid chromatography (FPLC) with anion exchange chromatography (AEC) or size exclusion chromatography (SEC) were adopted sequel to proteomics analysis. Separation of the cell extract of strain CSTR1 with SEC yielded active fractions with a molecular size range of 150-200 kDa. Conversely, using AEC resulted in significant loss of activity compared to SEC separation, and activities were majorly distributed in 3 complexes. Combinatorial experiment from AEC fractions show that the activity can be recovered when certain AEC fractions are combined but removal of a certain component resulted to a huge loss of activity up to 74%. This component that accounted for the majority of the nitrite reducing activity was denoted as an indispensable protein fraction. A hierarchical cluster analysis of the mass spectrometric identifications in the indispensable protein fraction revealed that a heme protein and electron transfer protein were linked to nitrite reducing activity. The evaluation of the physiological characteristics of the indispensable protein component revealed its ability to catalyse nitrite reduction in the presence of protein components such as AEC fractions, SEC fractions, reduced and oxidized haemoglobin, and bovine serum albumin (BSA). In contrast, these proteins without the indispensable protein component, as well as the indispensable protein component alone, exhibited no activity or significant loss of activity. Therefore, the discovery of the indispensable protein component in "Ca. Kuenenia stuttgartiensis" is an intriguing finding. These results suggest that protein interaction could be an important requirement for nitrite reduction in "Ca. Kuenenia stuttgartiensis".