The Role of Glutathione Mediated Oxidative Stress Response Mechanisms in Nitrifying Bacteria

Nitrification in activated sludge is especially susceptible to oxidative chemical toxins. Several oxidative stress response mechanisms exist in bacteria, and one highly conserved biomolecule involved with antioxidant activities is glutathione. In many Gram-negative heterotrophic bacteria, glutathione mediates the glutathione-gated potassium efflux (GGKE) response, which activates secondary stress responses that protect important intracellular components. A search of the genome of the Gram-negative ammonia oxidizing autotroph Nitrosomonas europaea revealed that glutathione synthase was present, while several key enzymes involved with glutathione reduction and GGKE are missing; however, other mechanisms that facilitate potassium efflux for oxidative stress protection may exist. Experiments were performed using the ammonia oxidizing bacterium N. europaea and the nitrite oxidizing bacterium Nitrospira moscoviensis. Pseudomonas aeruginosa PAO1 was used as a positive control. Concentrations of total and oxidized glutathione were measured in N. europaea after exposure to the oxidative chemical sodium hypochlorite. This data showed that glutathione was present but not oxidized to equivalent levels in N. europaea and P. aeruginosa. Additional batch experiments were performed and soluble potassium levels were monitored to observe increases associated with oxidant induced potassium efflux mechanisms. Results using N. europaea and Ni. moscoviensis suggest that neither bacteria effluxed potassium in response to the oxidants N-ethylmaleimide or sodium hypochlorite, indicating that no oxidative stress-induced potassium efflux mechanism exists in these nitrifiers. Coupled with known genomic information, the results of this study provide greater insight into why nitrification is so susceptible to process upset in biological wastewater treatment.

Biological wastewater treatment systems are the most common way to treat municipal wastewaters. Because municipal wastewater systems can receive wastewater from both domestic and industrial sources, accidental releases of shock loads of industrial chemical toxins can occasionally occur. These releases can upset the different treatment processes, like BOD removal and nitrification. These upset events can cause wastewater treatment facilities to violate discharge permits, which can lead to heavy fines or possible shutdown of the facility. Previous work done in our labs at Virginia Tech and by others has shown that conventional aerobic nitrification was the most sensitive biological wastewater treatment process to chemical inhibition (Kelly II et al., 2004; Blum and Speece, 1991; Tomlinson et al., 1966). From our experimental results, we determined that, when added to mixed liquor, each of 6 chemicals comprising different chemical classes inhibited nitrification, but the surrogate oxidative toxin was the most detrimental to nitrification. From this information, we decided to investigate why oxidative chemical toxins inhibit nitrification more severely than other chemical classes.

The process of nitrification is carried out by two distinct classes of autotrophic Gram-negative bacteria (Matin, 1978). The ammonia oxidizing bacteria (AOB) convert ammonia to nitrite (Hooper et al., 1997), while the nitrite oxidizing bacteria (NOB) convert nitrite to nitrate (Sundermeyer-Klinger et al., 1984), the end product of nitrification. Although oxidative chemicals are widely used in industry, few studies have been performed that examine the effects of these chemicals on activated sludge or nitrifying bacteria. Instead, much of the previous work has involved the use of pure cultures of Gram-negative bacteria. From these studies, it appears that the most likely inhibitory mechanisms in response to oxidative chemicals are through damaging DNA (Ferguson et al., 1996) and oxidizing thiol bonds and nucleophilic centers in proteins (Ferguson et al., 1997; McLaggan et al., 2000). Because DNA is essential to all living organisms, and several essential proteins in nitrifying bacteria contain thiol bonds at active sites (Meincke et al., 1992; Hooper et al.. 1997), these inhibitory mechanisms are likely causes of nitrification inhibition.

Unfortunately, only knowing how oxidative chemicals can inhibit nitrifying bacteria does not explain why they are more susceptible to inhibition by these chemicals. In order to determine what causes nitrifier susceptibility, it is important to examine the known bacterial stress response mechanisms for oxidative chemicals, or the molecular mechanisms bacteria have to help protect against oxidants. Recently, the complete genome of the AOB Nitrosomonas europaea was sequenced (Chain et al., 2003), allowing for a complete search of genes related to stress response and protection within this species. This search was completed and presented at the 2004 Water Environment Federation Technical Conference (Kelly and Love, 2004). Results of this search are summarized in Table 1, which lists several known oxidative stress response genes and whether these genes are present in N. europaea. From the list of stress response genes on Table 1, it appears that the genes encoding a majority of the protective enzymes are present in N. europaea, even though the protective enzymes that are present do not appear to have the same regulatory mechanisms as found in other bacteria. As presented previously (Kelly and Love, 2004), the only mechanism examined that does not appear to have enough genes to function properly is the glutathione gated potassium efflux (GGKE) mechanism first described by Kroll and Booth (1981). The loss or lack of this mechanism may help to explain the increased sensitivity of nitrifying bacteria to oxidative chemical toxins. Therefore, we decided to focus on the role of glutathione and potassium efflux mechanisms in protecting nitrifying bacteria that are exposed to oxidative chemical toxins.

The GGKE protection mechanism was first observed in pure cultures of the Gram-negative bacterium Escherichia coli (Kroll and Booth, 1981). This mechanism, which has been proposed to play a role in deflocculation events in activated sludge exposed to oxidative chemicals (Bott and Love, 2002; Bott and Love, 2004), appears to be highly conserved because it has been found in all Gram-negative heterotrophic species tested to date (Booth et al., 1993). As the name
implies, a very important molecule for activation of this stress response mechanism is glutathione. Glutathione is the major soluble non-protein thiol found in both eukaryotic and prokaryotic cells (Apontoweil and Berends, 1975) and provides a major cellular defense against oxidative compounds (Carmel-Harel and Storz, 2000; Ferguson et al., 2000; Ferguson and Booth, 1998). Glutathione helps protect cells from enzyme and DNA oxidation by acting as a “sacrificial lamb” because it scavenges oxidizing chemicals and is subsequently oxidized or conjugated with the chemical toxin (Vuilleumier, 1997). Once glutathione is oxidized it can usually be returned to its reduced state by a glutathione reductase (Carmel-Harel and Storz, 2000), which allows for glutathione to be recycled and reused within cells. Glutathione also protects cells by regulating the potassium efflux response in Gram-negative bacteria, as it activates the GGKE mechanism when it is oxidized. As the GGKE mechanism activates, potassium is released from the cells and the cytoplasm is acidified through import of protons. It is this acidification response that has been found to confer stress resistance to the cell (Ferguson et al., 2000; Ferguson and Booth, 1998; Ferguson et al., 1997; Ferguson et al., 1996).

After searching the genome of N. europaea for the genes that encode the proteins involved in the GGKE mechanism, several key absences were noted. A gene coding for glutathione reductase was not found (Chain et al., 2003), indicating that once the glutathione is oxidized it may not be able to be reduced for continued protection against oxidative chemicals. No matches were found for KefB and KefC (glutathione mediated K+/H+ antiporter channels), indicating that known GGKE-linked channels do not exist in N. europaea and the organism may not contain oxidantinduced cytoplasmic acidification mechanisms. In addition, Dps, a DNA protecting protein regulated by several systems including cytoplasmic pH (Ferguson et al., 1998), was absent from the N. europaea genome, further lending weight to the argument that acidification of the cytoplasm may not occur in nitrifying organisms. The absences of these mechanisms from the N. europaea genome indicate that the organism lacks a known GGKE mechanism and other essential oxidative stress protection mechanisms related to glutathione and GGKE. The lack of such mechanisms may explain why nitrifying bacteria are sensitive to electrophilic chemicals. As a genome sequence for an NOB has not yet been completed, it is unclear whether such mechanisms exist in NOB, indicating a need for further research.

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