A Novel Protein Protects Bacterial Iron-Dependent Metabolism from Nitric Oxide

Abstract

Reactive nitrogen species (RNS), in particular nitric oxide (NO), are toxic to bacteria, and bacteria have mechanisms to allow growth despite this stress. Understanding how bacteria interact with NO is essential to understanding bacterial physiology in many habitats, including pathogenesis; however, many targets of NO and enzymes involved in NO resistance remain uncharacterized. We performed for the first time a metabolomic screen on NO-treated and -untreated bacteria to define broadly the effects of NO on bacterial physiology, as well as to identify the function of NnrS, a previously uncharacterized enzyme involved in defense against NO. We found many known and novel targets of NO. We also found that iron-sulfur cluster enzymes were preferentially inhibited in a strain lacking NnrS due to the formation of iron-NO complexes. We then demonstrated that NnrS is particularly important for resistance to nitrosative stress under anaerobic conditions. Our data thus reveal the breadth of the toxic effects of NO on metabolism and identify the function of an important enzyme in alleviating this stress. Vibrio cholerae causes cholera, a severe watery diarrhea responsible for millions of cases and thousands of deaths each year (Centers for Disease Control and Prevention [http://www.cdc .gov]). It is not, however, a member of the Enterobacteriaceae-its natural habitat is aquatic. It is thought that during most of its life cycle, when not infecting humans, V. cholerae resides in association with zooplankton, forming biofilms on the chitinous surfaces of crustaceans (1, 2). Thus, V. cholerae must display metabolic flexibility in order to thrive in these two different environments and respond to the different metabolic challenges therein.A commonly encountered metabolic stress for pathogenic and nonpathogenic bacteria is the presence of reactive nitrogen species (RNS), in particular the well-studied molecule nitric oxide (NO). NO is formed as a by-product of nitrogen metabolism for many bacteria as an intermediate in denitrification (3), as well as from dedicated nitric oxide synthases (NOSs) in both bacteria and eukaryotes (4, 5), and is present in micromolar concentrations in some bacterial biofilms (6). NO can also be formed by chemical decomposition of nitrite in acid environments, such as the human stomach (7). NO is also a prominent component of the mammalian innate immune system, part of a battery of reactive oxygen species (ROS) and RNS produced by phagocytes when they encounter bacteria (7).The mechanisms whereby NO inhibits bacterial growth are diverse, but one of the most important properties of NO is its ability to bind iron and form dinitrosyl iron complexes (DNICs) bound to iron-sulfur cluster proteins, inhibiting their function (8, 9). DNICs have also been shown to mediate the formation of nitrosothiols, another form of nitrosative stress that inhibits thiolcontaining proteins (10). Through this mechanism and others, NO has been shown to inhibit a few enzymes in vitro, including such central metabolic enzymes as ...

Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

“…In addition, NO partitions to membranes (45). Hence, we suggest that NnrS may sense NO and signal to cytoplasmic transcription factors or Fe-S cluster repair proteins that result in the NO-protective effect previously observed (19).…”

“…This is consistent with work in Vibrio cholerae where an nnrS mutant showed hypersensitivity to NO under anaerobic conditions or in a ⌬hmpA (NO dioxygenase) background but showed no decrease in the NO degradation rate. There was also an increase in the formation of DNIC (dinitrosyl iron complex), the product of NO reacting with Fe-S clusters, indicating a connection with NO toxicity without direct removal of NO (19).…”

“…NirQ is proposed to work with NorD to generate active NorBC, although it is not known how the activation occurs (6). NnrS is a membrane-bound heme-Cu protein of unknown NOrelated function (18,19) that has not been characterized in gammaproteobacteria. Interestingly, NnrS and several other proteins connected with NO reduction, including NorBCD, are found in a variety of pathogens (see Fig.…”

Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide

“…Recently, a metabolomic screen to identify the effects of NO on the metabolism of V. cholerae revealed that NnrS is an NO-induced protein, which protects iron-sulfur proteins and the cellular iron-pool by lowering the production of dinitrosyliron complexes particularly under anaerobic conditions. 64 However, the best characterized NO detoxification systems are the enzymes flavohemoglobin (Hmp) and flavorubredoxin (NorV). 4 Hmp is primarily an NO dioxygenase, converting NO to NO 3 ¡ , although it has limited anoxic NO denitrosylase activity producing NO -(nitroxyl), which leads to the formation of N 2 O.…”

Role of norEF in Denitrification, Elucidated by Physiological Experiments with Rhodobacter sphaeroides

“…This is explained by the observation that norEF is not essential for Nor activity, but its absence does affect activity under conditions where endogenous Nir activity generates prolonged exposure to NO. This protein, along with NnrS, likely are representatives of a suite of proteins that permit robust denitrification but are not always found in partial denitrifiers (30). Therefore, they may be useful markers for active denitrification in metagenomic surveys in environments where denitrification is predicted to be an important physiological process (31).…”