Background: Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings. Results: Cellular DNIC formation upon brief nitric oxide ( ⅐ NO) exposure is dramatically increased by anoxia pretreatment. Conclusion: DNIC is rapidly generated from ⅐ NO reaction with chelatable iron. Significance: This experimental system provides a useful tool to manipulate the levels of DNIC and probe their cellular functions.
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 (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, 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 ΔnirS 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 ΔkatA mutant and a mucoid mucA22 ΔkatA 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 ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB 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 μM). 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.
This study compared SYBR Green real time quantitative polymerase chain reaction (qPCR) with standard plate counting for the enumeration of Streptococcus mutans in oral samples. Oral samples (N=710) were collected from high caries risk children for quantification of S. mutans using primer pairs by qPCR. S. mutans copy number (CN) was interpolated from qPCR quantification cycle (Cq) of samples compared to a S. mutans UA159 standard suspension. CN sample results were evaluated in relation to standard plate count (SPC) results obtained from each sample following culture on Petri plates using S. mutans selective media reported as colony forming units (CFU). Mean qPCR CN were found to be higher than SPC CFU (1.3 × 106 and 1.5 × 105, respectively). qPCR was usually higher in individual samples and qPCR detected the presence of S. mutans 84% (231/276) of time that SPC did not, compared to 33% (4/12) when qPCR failed to detect. qPCR was found to be more sensitive for detection of S. mutans from oral samples; a method that is not dependent on the viability of the sample taken and, therefore, is proposed as a more reliable and efficient means of quantification of S. mutans.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.