Figure 1 link
Denitrification has been widely reported in various taxa, and is especially diverse among proteobacterial groups (Zumft, 1997; Philippot, 2002); the utility of the pathway is underscored by the diversity of key constituent enzymes. The canonical denitrification enzyme is nitrite reductase, Nir, which reduces nitrite to nitric oxide. Nir functionality is found in two distinct enzymes—the copper-based nitrite reductase NirK, and the cytochrome-type reductase NirS (Brakeret al. , 2000; Priemé, Braker and Tiedje, 2002; Decleyre et al. , 2016).
The next step in the pathway—the reduction of nitric oxide to nitrous oxide—is catalyzed by nitric oxide reductases (NORs). Most bacterial NORs are homologous and closely related to one another, and to oxygen reductases in the heme-copper oxygen reductase superfamily (Hemp and Gennis, 2008). The most widely studied NOR enzymes are cNOR and qNOR (Hendriks et al. , 2000; Hemp and Gennis, 2008; Graf, Jones and Hallin, 2014), distinguished by their cytochrome or quinol electron donors, respectively. Rarer, alternative NOR enzymes, including sNOR, gNOR and eNOR, have been more recently identified and characterized in limited members of the Proteobacteria, Firmicutes, Archaea and Chloroflexi (Stein et al. , 2007; Hemp and Gennis, 2008; Sievertet al. , 2008; Hemp et al. , 2015).
Many bacteria contain only one or a partial subset of the four denitrification genes. Such organisms may perform partial denitrification, while others may use one of these enzymes for non-denitrifying functions (Hendriks et al. , 2000; Sanfordet al. , 2012; Graf, Jones and Hallin, 2014; Roco et al. , 2017). In partial denitrifers, the co-occurrence of denitrification pathway genes appears to vary across different taxa and environments (Graf, Jones and Hallin, 2014). Some of this variation may be constrained by the chemistry of certain intermediates. For example, nitric oxide (NO), the product of NirS and NirK, is highly cytotoxic. Both Nir types are periplasmic, and so cells require a means of effluxing or detoxifying nitric oxide before it accumulates to lethal levels. Denitrifiers are thought to immediately reduce nitric oxide to nitrous oxide to avoid injury, using membrane-bound NOR enzymes (Hendriks et al. , 2000). Perhaps for this reason, it is rare to find genomes that contain nir but not nor , while organisms showing the inverse—the presence of a nor gene but not anir gene—are far more common (Hendriks et al. , 2000; Graf, Jones and Hallin, 2014). While cNORs are only found in denitrifying microbes, other types of NOR—for example, quinol-dependent qNOR—are found in non-denitrifiers and can presumably detoxify environmental nitric oxides (Hendriks et al. , 2000). Beyond NORs, alternative pathways to nitric oxide detoxification are possible, including alternative enzymes such as cytochrome c oxidase (Blomberg and Ädelroth, 2018) or oxidoreductase (Gardner, Helmick and Gardner, 2002), flavorubredoxin (Gardner, Helmick and Gardner, 2002), or flavohemoglobins (Sánchez et al. , 2011).
While denitrification has been most widely studied and observed in Proteobacteria, the process has also been identified in other phyla, including Chloroflexi. Chloroflexi are ecologically and physiologically diverse, and often key players in oxygen-, nutrient-, and light-limited environments, including anaerobic sludge and subsurface sediments (Huget al. , 2013; Ward et al. , 2018). Previous surveys have indicated that, within Chloroflexi, members of Classes Chloroflexia and Thermomicrobia may have the capacity for nitrite reduction via the copper-type NirK (Wei et al. , 2015; Decleyre et al. , 2016). Recent studies indicate that certain Chloroflexi—especially members of Order Anaerolineales—may possess nirS instead ofnirK (Hemp et al. , 2015; Ward, McGlynn and Fischer, 2018), and may also harbor a divergent variant of nor previously reported in members of Archaea (Hemp and Gennis, 2008; Hemp et al. , 2015). These findings suggest that the evolution and/or biochemistry of denitrification may be unusual for this subset of bacteria, and informative for a broader understanding of microbial denitrification metabolisms and their origin.