Nitric Oxide Homeostasis in Salmonella typhimurium

Abstract

Nitric oxide (NO) is generated in biological systems primarily via the activity of NO synthases and nitrate and nitrite reductases. Here we show that Salmonella enterica serovar Typhimurium (S. typhimurium) grown anaerobically with nitrate is capable of generating polarographically detectable NO after nitrite (NO 2 ؊ ) addition. NO accumulation is sensitive to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Neither an fnr mutant nor an fnr hmp double mutant produces NO, indicating the involvement in NO evolution from NO 2 ؊ of protein(s) positively regulated by FNR. Contrary to previous findings in Escherichia coli, we demonstrate that neither the periplasmic nitrite reductase (NrfA) nor the cytoplasmic nitrite reductase (NirB) is involved in NO production in S. typhimurium. However, mutant cells lacking the membrane-bound nitrate reductase, NarGHI, and membranes derived from these cells are unable to produce NO, demonstrating that, in wild-type S. typhimurium, this enzyme is responsible for NO production. Membrane terminal oxidases cannot account for the NO levels measured. The nitrate reductase inhibitor, azide, abrogates NO evolution by Salmonella, and production of NO occurs only in the absence from the assays of nitrate; both features reveal a marked similarity between the NO-generating activities of this bacterium and plants. Unlike the situation in E. coli, an S. typhimurium hmp mutant produces NO both aerobically and anaerobically. Under aerobic conditions, when a functional flavohemoglobin is present, no NO is detectable. We propose a homeostatic mechanism in S. typhimurium, in which NO produced from NO 2 ؊ by nitrate reductase derepresses Hmp expression (via FNR and NsrR) and NorV expression (via NorR) and thus limits NO toxicity.

Origin and Impact of Nitric Oxide in Pseudomonas aeruginosa Biofilms

“…It is noteworthy that bacterial species devoid of nirK and nirS are also able to reduce nitrite to NO anaerobically; this capability likely involves products of fnr and hmp genes, as shown for Salmonella (74). Dispersal of Salmonella biofilms by NO donors was shown to depend on the presence of the recA-hydN genomic region, suggesting its involvement in NO sensing (75).…”

Compensations for Diminished Terminal Oxidase Activity in Escherichia coli

“…Respiration Assays-Samples were taken from cultures during mid-exponential growth (40 Klett), and harvested cells were washed with and resuspended in 50 mM Tris/HCl (pH 7.5); respiration was measured using a Clark-type polarographic oxygen electrode at 37°C (31). In brief, a 2-ml working volume was used, and respiration was initiated via injection of glycerol (5 mM final).…”

Novel flavohemoglobins of mycobacteria

“…Several putative functions have been proposed for flavoHbs (5–8). However, accumulating experimental evidences support its role in protection of microorganisms from the deleterious effects of nitric oxide (NO) by detoxifying it to nitrate via the NO-dioxygenation reaction (1, 2, 7, 8). The hydrophobic distal pocket of flavoHbs has been found much more flexible and expandable than conventional Hbs (3, 9) that allow them to accommodate large apolar molecules including phospholipids and perform other cellular functions.…”