Bacillus subtilis defends itself against the damaging effects of oxidative free-radicals by synthesizing nitric oxide.

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Immune cells like macrophages release free radicals when they encounter foreign microbes. Free radicals can cause oxidative stress including damage to DNA and cellular materials leading to cell death. Some bacteria, like Bacillus subtilis utilize a nitric oxide (NO) mediated system to rapidly respond to oxidative stress and minimize its effects. When reactive oxygen species (ROS) like hydrogen peroxide are detected by a bacterium, NO is quickly synthesized enzymatically. The NO counteracts the effects of the ROSs by two means. It activates a class of enzymes called catalases that break down hydrogen peroxide, or it starves the cell of the reduced form of iron needed by hydrogen peroxide to cause cell damage. This process increases bacterial resistance to hydrogen peroxide 100 fold in only 5 seconds. Although other processes like the synthesis of specialized proteins are responsible for long term resistance to ROSs, NO represents a critical rapid response chemical that confers immediate protection to the cell.

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"In bacteria, H2O2 [hydrogen peroxide] toxicity is attributable primarily to DNA damage. Upon interaction with free cellular iron, H2O2 forms hydroxyl radicals...that react at diffusion-limited rates with DNA bases and sugar moieties causing modifications and strand reduced iron, which is required for the Fenton reaction, is scarce in vivo and would be depleted almost instantaneously upon H2O2 challenge...rereduction of ferric ion by cellular reducing equivalents (RE) such as FADH2 and cysteine sustain the Fenton reaction, ultimately leading to cellular death...NO suppresses the Fenton reaction by transiently inhibiting cysteine reduction. Independently, NO specifically activates catalase to detoxify excess H2O2." (Gusarov and Nudler 2005:13855)

"NO Rapidly Protects B. subtilis from Oxidative Stress...within 5 sec of NO administration, resistance to H2O2 increased ~100-fold. The addition of NO simultaneously with or after H2O2 had no protective effect apparently because of NO scavenging by radicals generated by H2O2." (Gusarov and Nudler 2005:13856)

"NO has been shown to activate various genes in E. coli and B. subtilis to protect cells from oxidative and nitrosative stress. However, in our experiments the full protective effect of NO was established within 5 sec of NO administration, eliminating the necessity of gene activation for cytoprotection...the effect of a bolus of NO was transient, with a maximum attained within 5 sec of application. This finding is consistent with the short life time of NO in physiological solutions and argues for a rapid reversibility of the process. Taken together, these data suggest that NO directly and reversibly activates some latent, readily available oxidative stress defense system(s) in B. subtilis...NO could rapidly protect cells from H2O2 by boosting the activity of a preexisting H2O2 scavenging enzyme(s)...B. subtilis vegetative catalase KatA is the major antioxidant enzyme. It is an iron–heme protein, and thus a natural target for NO...Challenging a B. subtilis extract with our standard NO dose boosted H2O2 degradation by 75% but failed to do so in an extract from ΔkatA cells, demonstrating that NO indeed potentiates the activity of KatA...NO relieves this inhibition by disrupting the KatA–Cys complex apparently via an S-nitrosylation mechanism...DNA damage from hydroxyl radicals generated by the Fenton reaction is a primary mechanism of H2O2 cytotoxicity...NO protection occurs via the inhibition of Fenton chemistry." (Gusarov and Nubler 2005:13857)

"H2O2 oxidizes cellular iron to yield highly toxic hydroxyl radicals. Cys reduces iron back again by forming cystine. Trx/TrxRed then reduces cystine back to Cys at the expense of NADPH. To keep the Fenton reaction going, oxidized iron and cystine must be continuously rereduced...NO transiently interrupts cystine reduction by inhibiting Trx/TrxRed, thus rendering the Fenton reaction non- processive. Simultaneously, NO directly activates catalase, which detoxifies excess H2O2." (Gusarov and Nubler 2005:13859)

Journal article
Bacterial Nitric Oxide SynthasesAnnu. Rev. Biochem.June 9, 2010
Brian R. Crane, Jawahar Sudhamsu, Bhumit A. Patel

Journal article
NO-mediated cytoprotection: Instant adaptation to oxidative stress in bacteriaProceedings of the National Academy of SciencesSeptember 20, 2005
I. Gusarov, E. Nudler

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