A combination of cytochrome c nitrite reductase (NrfA) and flavorubredoxin (NorV) protects Salmonella enterica serovar Typhimurium against killing by NO in anoxic environments.
The enteric bacterium Salmonella enterica serovar Typhimurium is a pathogen that is highly adapted for both intracellular and extracellular survival in a range of oxic and anoxic environments. The cytotoxic radical nitric oxide (NO) is encountered in many of these environments. Protection against NO may involve reductive detoxification in low-oxygen environments, and three enzymes, flavorubredoxin (NorV), flavohaemoglobin (HmpA) and cytochrome c nitrite reductase (NrfA), have been shown to reduce NO in vitro. In this work we determined the role of these three enzymes in NO detoxification by Salmonella by assessing the effects of all eight possible combinations of norV, hmpA and nrfA single, double and triple mutations. The mutant strains were cultured and exposed to NO following either glucose fermentation (when nitrite reductase activity is low), or anaerobic respiration (when nitrite reductase activity is high). Wild-type cultures were more sensitive to the addition of a pulse of NO when grown under fermentative conditions compared with anaerobic respiratory conditions. Analysis of the mutant strains suggested an important additive role for both NorV and NrfA in both environments, since the norV nrfA mutant could not grow after NO addition. The results also suggested a minor role for HmpA in anaerobic detoxification of NO under the two growth conditions, and a larger role for HmpA in aerobic NO detoxification was confirmed. Activity assays and measurements of NO consumption showed that increased nitrite reductase activity correlates with an elevated capacity for NO reduction by intact cells. Taken together, the results reveal a combined role for NorV and NrfA in NO detoxification under anaerobic conditions, and highlight the influence that growth conditions have on the sensitivity to NO of this pathogenic bacterium.
Mills
Paul C
PC
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Rowley
Gary
G
Molecular Microbiology Group, Institute of Food Research, Norwich NR4 7UA, UK.
Spiro
Stephen
S
Department of Molecular and Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX 75080, USA.
Hinton
Jay C D
JCD
Molecular Microbiology Group, Institute of Food Research, Norwich NR4 7UA, UK.
Richardson
David J
DJ
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
D000693:Anaerobiosis; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D045263:Cytochromes a1 / Q000235:genetics / Q000378:metabolism*; D003575:Cytochromes c1 / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism; D050296:Microbial Viability; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D009565:Nitrate Reductases / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D012416:Rubredoxins / Q000235:genetics / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000254:growth & development / Q000502:physiology*
D000900:Anti-Bacterial Agents; D012416:Rubredoxins; D009569:Nitric Oxide; D045263:Cytochromes a1; D003575:Cytochromes c1; D009247:NADH, NADPH Oxidoreductases; D009565:Nitrate Reductases; C054690:nitrate reductase (cytochrome); D005947:Glucose