Many gram-negative bacteria harbor a copper/zinc-containing superoxide dismutase (CuZnSOD) in their periplasms. In pathogenic bacteria, one role of this enzyme may be to protect periplasmic biomolecules from superoxide that is released by host phagocytic cells. However, the enzyme is also present in many nonpathogens and/or free-living bacteria, including Escherichia coli. In this study we were able to detect superoxide being released into the medium from growing cultures of E. coli. Exponential-phase cells do not normally synthesize CuZnSOD, which is specifically induced in stationary phase. However, the engineered expression of CuZnSOD in growing cells eliminated superoxide release, confirming that this superoxide was formed within the periplasm. The rate of periplasmic superoxide production was surprisingly high and approximated the estimated rate of cytoplasmic superoxide formation when both were normalized to the volume of the compartment. The rate increased in proportion to oxygen concentration, suggesting that the superoxide is generated by the adventitious oxidation of an electron carrier. Mutations that eliminated menaquinone synthesis eradicated the superoxide formation, while mutations in genes encoding respiratory complexes affected it only insofar as they are likely to affect the redox state of menaquinone. We infer that the adventitious autoxidation of dihydromenaquinone in the cytoplasmic membrane releases a steady flux of superoxide into the periplasm of E. coli. This endogenous superoxide may create oxidative stress in that compartment and be a primary substrate of CuZnSOD.

SummaryMechanisms of hydrogen peroxide generation in Escherichia coli were investigated using a strain lacking scavenging enzymes. Surprisingly, the deletion of many abundant flavoenzymes that are known to autoxidize in vitro did not substantially lessen overall H2O2 formation. However, H2O2 production diminished by 25-30% when NadB turnover was eliminated. The flavin-dependent desaturating dehydrogenase, NadB uses fumarate as an electron acceptor in anaerobic cells. Experiments showed that aerobic NadB turnover depends upon its oxidation by molecular oxygen, with H2O2 as a product. This reaction appears to be mechanistically adventitious. In contrast, most desaturating dehydrogenases are associated with the respiratory chain and deliver electrons to fumarate anaerobically or oxygen aerobically without the formation of toxic by-products. Presumably, NadB can persist as an H2O2-generating enzyme because its flux is limited. The anaerobic respiratory enzyme fumarate reductase uses a flavoprotein subunit that is homologous to NadB and accordingly forms substantial H2O2 upon aeration. This tendency is substantially suppressed by cytochrome oxidase. Thus cytochrome d oxidase, which is prevalent among anaerobes, may diminish intracellular H2O2 formation by the anaerobic respiratory chain, whenever these organisms encounter oxygen. These two examples reveal biochemical and physiological arrangements through which evolution has minimized the rate of intracellular oxidant formation.

, [95][96][97][98][99][100][101][102][103][104][105][106]

The material balance method, being the basic algorithm of leak detection systems (LDS), is discussed. A criterion for making a decision concerning a leak is substantiated; the issue of determining the sensitivity limit of the material balance algorithm is discussed.

This paper discusses the method of hydraulic location for leak detection. A statistical criterion for leakage incident detection has been developed. A series of simulation experiments conducted has confirmed that the developed criteria are adequate. An algorithm has been developed to evaluate the sensitivity of a leak detection algorithm based on the availability of pressure instruments at the process section.