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How does air block glucose metabolism in the anaerobic bacterium Bacterium thetaiotaomicron?
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345
DETECTION OF OXIDATIVE STRESS INDUCTION USING GREEN FLUORESCENT PROTEIN PROMOTER PROBES $“Zi;~~o~.~-Eichha~, W.E.MtC and GouindRno.
PSEUDO - CATALATIC MODE OF HORSE RADISH PEROXIDASE
Transcriptional fusions using reporter genes have long been used to study the induction of oxklative stress genes. We have used this proven approach to employ the relatively new reporter gene, green fluorescent protein (GFPJ. Several GFP promoter probes have been constructed using genes within the oxldative stress re ons. The promoters of sodA, zwf, acnA, dpe, dnaK, kntE, an p recA were amplified by PCR and cloned upstream of GFP. These GFP remoter probes transformed into Escherlchla coli were ex & to various stresses. Activation of the promoter results ln fluorescence intensity emission by GPP which is easily detected and quantitated. The GFP promoter probes within the SoxRS regulon show speclflc dose dependent responses when stressed with paraquat but not with hydrogen peroxide. In contrast, those GFP promoter probes wlthln the OxyR regulon and SOS response show a specific dose dependent response to hydrogen peroxide and minimally with paraquat. In addition to testing the GFP promoter probes with known oxldative stresses, we also present data using hyperoxia. Thll experimental design establishes a paradlgm for rapid, non-invasive, and quantitative assessment of oxldative stress induction.
Peroxidases are complex enzymes that serve many functions in plants. In addition to consuming hydrogen peroxide in the oxidation of various substrates (peroxtdatlve mode), they also are able to produce hydrogen peroxide via utilization of extracellular NASH (pseudo-oxkiative mode). The current study describes a third type of peroxldase activity, a “pseudo-catalatic” mechanism that apparently produces molecular oxygen at the expense of hydrogen peroxide. Both the oxygen production and hydrogen peroxide - scavenging activity were vulnerable to changes in temperature and had fairly broad pH optima ln the neutral to alkaline region. The apparent Km of the oxygen production and hydrogen peroxide-scavenging reactions were ln the range of 1.OmM. Irreversible lnactlvatlon of peroxkiase by exposure to high concentrations of hydrogen peroxide colnclded with the formation of an absorbance peak at 670 run. Unlike true catalase activity, SOD enhanced and gualacol inhibited the pseudo-catalatic activity of peroxidase. It appears that peroxldase is capable of using hydrogen peroxide as an electron donor in the absence of other substrates. The superoxlde produced appears to either d&mutate or serve as an electron donor for peroxkiase producing molecular oxygen.
in the thetaiotaomicron?
How Does Air Block Glucose Metabolism
Anaerobic
Bacterium
Bacteroides
&&,!a~ and James A. halay Dept. of Microbiology, Univ. of’Illinois. Urbana, IL 61801 It is often unclear why obligate anaerobes caonoc grow in air. In 1971 McCord. Keele, and Fridovich proposed that soperoxide anion (0~) may be responsible. Bacteroides rhelaioraomicron is a gram-negativeobligately anaerobic bacterium found in large numbers in the homan colon. When B. rheraioraomicron is shifted from an anaerobic to an aerobic environment,
glucose metabolismslows and the cells stop growing. The biomolecules knownto be most vulnerableto oxygensaess are dehydmraseswhoseircnsulfur clusters are rapidly damaged by Or. Famamse. which bacteria is ao iron-sulfur cluster dehydratase, is required for We hypothesized that metabolism in B. rheraioraomicron. metabolism may be blocked by air because fmnarase is damaged by
ia some glucose glucose 0~.
B. the~aiotaomicron generates py~vate, lactate, acetate, succinate. and propionate when it ferments glucose anaerobically. ‘H Nh4R analysis showed that air-exposed cells continue to make pymvate, lactate, and xetate. However, the formation of succinate and pmpionate,which requires fumamse activity and is essential for growth, stopped. Succinate was produced by aerobic cultures if fomamte was provided exogenously, confaming that the block lies in the conversion of pyruvate to fomarate. In vitro analysis showed that the B. rheraioruomicron fmnamse can be inxtiviued rapidly by 0~ and oxygen. ‘This verified that fmnarase contains a labile iron-sulfur cluster. Furthermore, famarase activity rapidly decreased when cells were exposed to oxygen, while other enzymes were still active. This inactivation could be protected by expressing E. coli sods gene in B. dae~aioraomicron. These results suggest that fmnamse inactivation by Oz- is a key factor when air blocks glucose metabolism. Therefore we are testing whether the transgenic expression of a superoxide-resistant fmnarase from E. coli will enable the obligate anzmbe to ferment sugars in air.
OXYGEN
’ 9
8
Sl21
345
DETECTION OF OXIDATIVE STRESS INDUCTION USING GREEN FLUORESCENT PROTEIN PROMOTER PROBES $“Zi;~~o~.~-Eichha~, W.E.MtC and GouindRno.
PSEUDO - CATALATIC MODE OF HORSE RADISH PEROXIDASE
Transcriptional fusions using reporter genes have long been used to study the induction of oxklative stress genes. We have used this proven approach to employ the relatively new reporter gene, green fluorescent protein (GFPJ. Several GFP promoter probes have been constructed using genes within the oxldative stress re ons. The promoters of sodA, zwf, acnA, dpe, dnaK, kntE, an p recA were amplified by PCR and cloned upstream of GFP. These GFP remoter probes transformed into Escherlchla coli were ex & to various stresses. Activation of the promoter results ln fluorescence intensity emission by GPP which is easily detected and quantitated. The GFP promoter probes within the SoxRS regulon show speclflc dose dependent responses when stressed with paraquat but not with hydrogen peroxide. In contrast, those GFP promoter probes wlthln the OxyR regulon and SOS response show a specific dose dependent response to hydrogen peroxide and minimally with paraquat. In addition to testing the GFP promoter probes with known oxldative stresses, we also present data using hyperoxia. Thll experimental design establishes a paradlgm for rapid, non-invasive, and quantitative assessment of oxldative stress induction.
Peroxidases are complex enzymes that serve many functions in plants. In addition to consuming hydrogen peroxide in the oxidation of various substrates (peroxtdatlve mode), they also are able to produce hydrogen peroxide via utilization of extracellular NASH (pseudo-oxkiative mode). The current study describes a third type of peroxldase activity, a “pseudo-catalatic” mechanism that apparently produces molecular oxygen at the expense of hydrogen peroxide. Both the oxygen production and hydrogen peroxide - scavenging activity were vulnerable to changes in temperature and had fairly broad pH optima ln the neutral to alkaline region. The apparent Km of the oxygen production and hydrogen peroxide-scavenging reactions were ln the range of 1.OmM. Irreversible lnactlvatlon of peroxkiase by exposure to high concentrations of hydrogen peroxide colnclded with the formation of an absorbance peak at 670 run. Unlike true catalase activity, SOD enhanced and gualacol inhibited the pseudo-catalatic activity of peroxidase. It appears that peroxldase is capable of using hydrogen peroxide as an electron donor in the absence of other substrates. The superoxlde produced appears to either d&mutate or serve as an electron donor for peroxkiase producing molecular oxygen.
in the thetaiotaomicron?
How Does Air Block Glucose Metabolism
Anaerobic
Bacterium
Bacteroides
&&,!a~ and James A. halay Dept. of Microbiology, Univ. of’Illinois. Urbana, IL 61801 It is often unclear why obligate anaerobes caonoc grow in air. In 1971 McCord. Keele, and Fridovich proposed that soperoxide anion (0~) may be responsible. Bacteroides rhelaioraomicron is a gram-negativeobligately anaerobic bacterium found in large numbers in the homan colon. When B. rheraioraomicron is shifted from an anaerobic to an aerobic environment,
glucose metabolismslows and the cells stop growing. The biomolecules knownto be most vulnerableto oxygensaess are dehydmraseswhoseircnsulfur clusters are rapidly damaged by Or. Famamse. which bacteria is ao iron-sulfur cluster dehydratase, is required for We hypothesized that metabolism in B. rheraioraomicron. metabolism may be blocked by air because fmnarase is damaged by
ia some glucose glucose 0~.
B. the~aiotaomicron generates py~vate, lactate, acetate, succinate. and propionate when it ferments glucose anaerobically. ‘H Nh4R analysis showed that air-exposed cells continue to make pymvate, lactate, and xetate. However, the formation of succinate and pmpionate,which requires fumamse activity and is essential for growth, stopped. Succinate was produced by aerobic cultures if fomamte was provided exogenously, confaming that the block lies in the conversion of pyruvate to fomarate. In vitro analysis showed that the B. rheraioruomicron fmnamse can be inxtiviued rapidly by 0~ and oxygen. ‘This verified that fmnarase contains a labile iron-sulfur cluster. Furthermore, famarase activity rapidly decreased when cells were exposed to oxygen, while other enzymes were still active. This inactivation could be protected by expressing E. coli sods gene in B. dae~aioraomicron. These results suggest that fmnamse inactivation by Oz- is a key factor when air blocks glucose metabolism. Therefore we are testing whether the transgenic expression of a superoxide-resistant fmnarase from E. coli will enable the obligate anzmbe to ferment sugars in air.
OXYGEN
’ 9
8
Sl21