ABSTRACTNitrite has been used as a bacteriostatic agent for centuries in food preservation. It is widely accepted that this biologically inert molecule functions indirectly, serving as a stable reservoir of bioactive nitric oxide (NO) and other reactive nitrogen species to impact physiology. As a result, to date, we know surprisingly little aboutin vivotargets of nitrite. Here, we carry out comparative analyses of nitrite and NO physiology inEscherichia coliand inShewanella oneidensis, a Gram-negative environmental bacterium renowned for respiratory versatility. These two bacteria differ from each other in many aspects of nitrite and NO physiology, including NO generation, NO degradation, and unexpectedly, their contrary susceptibility to nitrite and NO. In cell extracts of both bacteria, most of the NO targets are also susceptible to nitrite, and vice versa. However, with respect to growth inhibition caused by NO, the targets are impacted distinctly; NO targets are responsible for the inhibition of growth ofE. colibut not ofS. oneidensis. More surprisingly, all proteins identified to be implicated in NO tolerance in other bacteria appear to play a dispensable role in protectingS. oneidensisagainst NO. These data suggest thatS. oneidensisis equipped with a robust but yet unknown NO protecting system. In the case of nitrite, it is clear that the target of physiological significance in both bacteria is cytochrome heme-copper oxidase.IMPORTANCENitrite is toxic to living organisms at high levels, but such antibacterial effects of nitrite are attributable to the formation of nitric oxide (NO), a highly reactive radical gas molecule. Here, we report thatShewanella oneidensisis highly resistant to NO but sensitive to nitrite compared toEscherichia coliby approximately 4-fold. In both bacteria, nitrite inhibits bacterial growth by targeting cytochrome heme-copper oxidase. In contrast, the targets of NO are diverse. Although these targets are similar inE. coliandS. oneidensis, they are responsible for growth inhibition caused by NO in the former but not in the latter. Overall, the presented data, along with the previous data, solidify a proposal that thein vivotargets of NO and nitrite in bacteria are largely different.
Structural basis for nitrous oxide generation by bacterial nitric oxide reductases