Nitrite Derived from Endogenous Bacterial Nitric Oxide Synthase Activity Promotes Aerobic Respiration (original) (raw)
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Molecular microbiology, 2017
Nitric oxide (NO) is generated from arginine and oxygen via NO synthase (NOS). Staphylococcus aureus NOS (saNOS) has previously been shown to affect virulence and resistance to exogenous oxidative stress, yet the exact mechanism is unknown. Herein, a previously undescribed role of saNOS in S. aureus aerobic physiology was reported. Specifically, aerobic S. aureus nos mutant cultures presented with elevated endogenous reactive oxygen species (ROS) and superoxide levels, as well as increased membrane potential, increased respiratory dehydrogenase activity and slightly elevated oxygen consumption. Elevated ROS levels in the nos mutant likely resulted from altered respiratory function, as inhibition of NADH dehydrogenase brought ROS levels back to wild-type levels. These results indicate that, in addition to its recently reported role in regulating the switch to nitrate-based respiration during low-oxygen growth, saNOS also plays a modulatory role during aerobic respiration. Multiple tr...
Functional and Mechanistic Characterization of Bacterial Nitric Oxide Signaling Pathways
2016
Author(s): Rao, Minxi | Advisor(s): Marletta, Michael A. | Abstract: Nitric oxide (NO) is a well-established signaling molecule and cytotoxic agent in mammals. NO is synthesized by nitric oxide synthase (NOS) by macrophages at high concentrations as a key part of the host immune response, and at low concentrations in endothelial and neuronal cells as a signaling agent. In endothelial cells, the primary NO receptor is soluble guanylate cyclase (sGC), which contains a heme-nitric oxide/oxygen binding domain (H-NOX). Selective binding of NO to the H-NOX domain is responsible for activation of sGC. Thus, the mammalian NO signaling system involves NO synthesis by NOS, and NO sensing by the H-NOX domain of sGC. NOS and H-NOX proteins have also been identified in a number of bacterial species, including pathogens. Putative roles for bacterial NOS proteins include protection against oxidative stress and antibiotics, while bacterial H-NOX proteins have been shown to govern processes such as ...
Molecular Microbiology, 2015
Staphylococcus aureus nitrosative stress resistance is due in part to flavohemoprotein (Hmp). Although hmp is present in all sequenced S. aureus genomes, 37% of analyzed strains also contain nor, encoding a predicted quinol-type NO reductase (saNOR). DAF-FM staining of NOchallenged wild-type, nor, hmp, and nor hmp mutant biofilms suggested that Hmp may have a greater contribution to intracellular NO detoxification relative to saNOR. However, saNOR still had a significant impact on intracellular NO levels, and complemented NO detoxification in a nor hmp mutant. When grown as NO-challenged static (low-oxygen) cultures, hmp and nor hmp mutants both experienced a delay in growth initiation, whereas the nor mutant's ability to initiate growth was comparable to the wild-type strain. However, saNOR contributed to cell respiration in this assay once growth had resumed, as determined by membrane potential and respiratory activity assays. Expression of nor was upregulated during low-oxygen growth and dependent on SrrAB, a two-component system that regulates expression of respiration and nitrosative stress resistance genes. High-level nor promoter activity was also detectable in a cell subpopulation near the biofilm substratum. These results suggest that saNOR contributes to NO-dependent respiration during nitrosative stress, possibly conferring an advantage to nor+ strains in vivo.
The Staphylococcus aureus α-Acetolactate Synthase ALS Confers Resistance to Nitrosative Stress
Frontiers in microbiology, 2017
Staphylococcus aureus is a worldwide pathogen that colonizes the human nasal cavity and is a major cause of respiratory and cutaneous infections. In the nasal cavity, S. aureus thrives with high concentrations of nitric oxide (NO) produced by the innate immune effectors and has available for growth slow-metabolizing free hexoses, such as galactose. Here, we have used deep sequencing transcriptomic analysis (RNA-Seq) and (1)H-NMR to uncover how S. aureus grown on galactose, a major carbon source present in the nasopharynx, survives the deleterious action of NO. We observed that, like on glucose, S. aureus withstands high concentrations of NO when using galactose. Data indicate that this resistance is, most likely, achieved through a distinct metabolism that relies on the increased production of amino acids, such as glutamate, threonine, and branched-chain amino acids (BCAAs). Moreover, we found that under NO stress the S. aureus α-acetolactate synthase (ALS) enzyme, which converts py...
Nitrogen oxide cycle regulates nitric oxide levels and bacterial cell signaling
Scientific Reports, 2016
Nitric oxide (NO) signaling controls various metabolic pathways in bacteria and higher eukaryotes. Cellular enzymes synthesize and detoxify NO; however, a mechanism that controls its cellular homeostasis has not been identified. Here, we found a nitrogen oxide cycle involving nitrate reductase (Nar) and the NO dioxygenase flavohemoglobin (Fhb), that facilitate inter-conversion of nitrate, nitrite, and NO in the actinobacterium Streptomyces coelicolor. This cycle regulates cellular NO levels, bacterial antibiotic production, and morphological differentiation. NO down-regulates Nar and up-regulates Fhb gene expression via the NO-dependent transcriptional factors DevSR and NsrR, respectively, which are involved in the auto-regulation mechanism of intracellular NO levels. Nitrite generated by the NO cycles induces gene expression in neighboring cells, indicating an additional role of the cycle as a producer of a transmittable inter-cellular communication molecule. Nitric oxide (NO) is a freely diffusible neutral gas that acts as an important signaling molecule to control metabolic pathways in bacteria and higher eukaryotes. Since NO is highly reactive and toxic for living cells, cells must have strict control over intracellular NO levels. The genus Streptomyces includes bacteria that produce many commercially useful secondary metabolites that are extremely important to humans. They follow an elaborate life cycle that includes vegetative (or substrate) mycelial growth, aerial mycelial growth, and sporulation. Recent studies suggest that actinobacteria require NO to regulate various metabolic pathways 1-5 , however, little is known about the mechanism by which actinobacteria generate NO, except for NO synthase (NOS) distribution in a limited number of actinobacterial species. Recently, we reported a unique nitrogen metabolism in Streptomyces antibioticus, in which organic nitrogen was mineralized to form nitrogen oxide species, nitrite (NO 2 −), nitrate (NO 3 −), and NO during vegetative cell growth under aerobic conditions, and NO 2 − was excreted into the medium 6. Since arginine analogs inhibited the production of NO 2 − and NO, we suggested that NOS is involved in the NO 2 −-formation, and proposed a NO 2 −-forming pathway (pathway 1, below), although presence of a NOS enzyme has not been demonstrated in S. antibioticus.
The EMBO Journal, 2007
Protection from NO gas, a toxic byproduct of anaerobic respiration in Pseudomonas aeruginosa, is mediated by nitric oxide (NO) reductase (NOR), the norCB gene product. Nevertheless, a norCB mutant that accumulated B13.6 lM NO paradoxically survived anaerobic growth. Transcription of genes encoding nitrate and nitrite reductases, the enzymes responsible for NO production, was reduced 450and 2.5-fold in the norCB mutant. This was due, in part, to a predicted compromise of the [4Fe-4S] 2 þ cluster in the anaerobic regulator ANR by physiological NO levels, resulting in an inability to bind to its cognate promoter DNA sequences. Remarkably, two O 2-dependent dioxygenases, homogentisate-1,2-dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (Hpd), were derepressed in the norCB mutant. Electron paramagnetic resonance studies showed that HmgA and Hpd bound NO avidly, and helped protect the norCB mutant in anaerobic biofilms. These data suggest that protection of a P. aeruginosa norCB mutant against anaerobic NO toxicity occurs by both control of NO supply and reassignment of metabolic enzymes to the task of NO sequestration.
The Response of nor and nos Contributes to Staphylococcus aureus Virulence and Metabolism
Journal of Bacteriology
Staphylococcus aureus causes a wide spectrum of disease, with the site and severity of infection dependent on virulence traits encoded within genetically distinct clonal complexes (CCs) and bacterial responses to host innate immunity. The production of nitric oxide (NO) by activated phagocytes is a major host response to which S. aureus metabolically adapts through multiple strategies that are conserved in all CCs, including an S. aureus nitric oxide synthase (Nos). Previous genome analysis of CC30, a lineage associated with chronic endocardial and osteoarticular infections, revealed a putative NO reductase (Nor) not found in other CCs that potentially contributes to NO resistance and clinical outcome. Here, we demonstrate that Nor has true nitric oxide reductase activity, with nor expression enhanced by NO stress and anaerobic growth. Furthermore, we demonstrate that nor is regulated by MgrA and SrrAB, which modulate S. aureus virulence and hypoxic response. Transcriptome analysis ...
Catalase (KatA) Plays a Role in Protection against Anaerobic Nitric Oxide in Pseudomonas aeruginosa
PLoS ONE, 2014
Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H 2 O 2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H 2 O 2 , is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant DnirS produced ,50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A DkatA mutant and a mucoid mucA22 DkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the DkatA mutant, and dramatically in a DnorCB mutant compared to basal levels of DNIC in PAO1 and DnirS mutant. Expression of KatA dramatically reduced the DNIC levels in DnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (K d ,6 mM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.
Molecular microbiology, 2005
The human pathogen Neisseria meningitidis is the major causative agent of bacterial meningitis. The organism is usually treated as a strict aerobe and is cultured under fully aerobic conditions in the laboratory. We demonstrate here that although N. meningitidis fails to grow under strictly anaerobic conditions, under oxygen limitation the bacterium expresses a denitrification pathway (reduction of nitrite to nitrous oxide via nitric oxide) and that this pathway supplements growth. The expression of the gene aniA, which encodes nitrite reductase, is regulated by oxygen depletion and nitrite availability via transcriptional regulator FNR and two-component sensor-regulator NarQ/NarP respectively. Completion of the two-step denitrification pathway requires nitric oxide (NO) reduction, which proceeds after NO has accumulated during batch growth under oxygen-limited conditions. During periods of NO accumulation both nitrite and NO reduction are observed aerobically, indicating N. meningi...