ESR DETECTION OF NITRIC OXIDE PRODUCED IN VIVO BY PROKARYOTIC CELLS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Prof Rosemary Loria is interested in detection, via ESR, of nitric oxide produced in vivo by prokaryotic cells. Nitric oxide synthases (NOSs) in mammals are dimeric proteins containing N-terminal oxygenase domains that convert arginine into citrulline and NO, and a reductase domain that shuttles electrons to the heme of the oxygenase domain. Homologs to NOSs exist in prokaryots, but direct evidence for NO production by these proteins has been lacking. The aim of current work was to demonstrate that NOS also produces diffusible NO. To demonstrate NO production, the spin trap dithiocarboxy-sarcosine (DTCS) was used. DTCS binds iron to form the active spin trap Fe(DTCS)2 that lacks an ESR signal. After NO binds to the Fe(DTCS)2 complex, the spin trap produces a characteristic 3-peak signal. Incubation of Fe(DTCS)2 with the wild type culture of S. turgidiscabies resulted in the appearance of the characteristic 3-peak signal of NO-Fe(DTCS)2 identical to that produced in control experiments by addition of artificial NO-donor. This NO-specific 3-peak signal was not observed in samples from NOS-deletion mutant cultures. These results confirmed that NOS-dependent free-diffusing NO is released by thaxtomin-producing streptomycetes and were the first data to document in vivo production of NO by prokaryotic NOSs. Structural conservation of bacterial NOSs predicts NO production by other Gram-positive bacteria including medically and environmentally important organisms such as Bacillus anthracis, Staphylococcus aureus, and Deinococcus radiodurans. A paper on this work was submitted to Chemistry & Biology. This work also represents a substantial part of the PhD thesis of a graduate student of Prof. Loria: Evan Johnson.