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Redox analysis of the cytochrome o-type quinol oxidase complex of Escherichia coli reveals three redox components.

ABSTRACT: Potentiometric analyses of the cytochrome o-type oxidase of Escherichia coli, using membranes from a strain containing amplified levels of the cytochrome bo complex, were conducted to resolve the redox centres of the oxidase. The cytochrome o-type oxidase of E. coli, a quinol oxidase, contains 2 mol of b-type haem per mol of complex and copper. Detailed analysis of potentiometric titrations, based on the absorbance of the Soret band, suggests that there are three contributions with midpoint potentials (Em,7) around +55 mV, +211 mV and +408 mV, all with maxima at 426-430 nm in the reduced state. In the alpha region of the spectra, a component with Em,6.85 = +58 mV has a maximal peak at 557 nm, and twin peaks at 556 and 564 nm nitrate with Em,6.85 = +227 mV. A feature corresponding to the highest potential Soret contribution was not observed. These data can be explained either by a model incorporating haem-haem interaction or by attributing the shorter-wavelength band (557 nm) to haem b and a split alpha-band (556, 564 nm) to the haem o (oxygen-binding haem b). Absolute spectra of oxidized membranes show continuous absorbance from 460 to 530 nm and suggest the presence of a high-spin haem component in the membranes. Monitoring absorbance at 635 minus 672 nm, contributions with midpoints (Em,7) around +52 mV, +234 mV and +371 mV are observed. This latter contribution is possibly the highest-potential component which titrates with Em greater than +400 mV in the Soret region and may represent copper-haem coupling in the cytochrome o complex.

SUBMITTER: Bolgiano B

PROVIDER: S-EPMC1149971 | biostudies-other | 1991 Mar

REPOSITORIES: biostudies-other

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Project description:A hallmark of bacterial biofilms is the production of an extracellular matrix (ECM) that encases and protects the community from environmental stressors. Biofilm formation is an integral portion of the uropathogenic Escherichia coli (UPEC) life cycle. Approximately 2% of UPEC isolates are cysteine auxotrophs. Here, we investigated how cysteine homeostasis impacted UPEC UTI89 strain biofilm formation and, specifically, the production of the ECM components curli and cellulose. Cysteine auxotrophs produced less cellulose and slightly more curli compared to wild-type (WT) strains, and cysteine auxotrophs formed smooth, nonrugose colonies. Cellulose production was restored in cysteine auxotrophs when YfiR was inactivated. YfiR is a redox-sensitive regulator of the diguanylate cyclase, YfiN. The production of curli, a temperature-regulated appendage, was independent of temperature in UTI89 cysteine auxotrophs. In a screen of UPEC isolates, we found that ∼60% of UPEC cysteine auxotrophs produced curli at 37°C, but only ∼2% of cysteine prototrophic UPEC isolates produced curli at 37°C. Interestingly, sublethal concentrations of amdinocillin and trimethoprim-sulfamethoxazole inhibited curli production, whereas strains auxotrophic for cysteine continued to produce curli even in the presence of amdinocillin and trimethoprim-sulfamethoxazole. The dysregulation of ECM components and resistance to amdinocillin in cysteine auxotrophs may be linked to hyperoxidation, since the addition of exogenous cysteine or glutathione restored WT biofilm phenotypes to mutants unable to produce cysteine and glutathione.IMPORTANCE Uropathogenic Escherichia coli (UPEC) bacteria are the predominant causative agent of urinary tract infections (UTIs). UTIs account for billions of dollars of financial burden annually to the health care industry in the United States. Biofilms are an important aspect of the UPEC pathogenesis cascade and for the establishment of chronic infections. Approximately 2% of UPEC isolates from UTIs are cysteine auxotrophs, yet there is relatively little known about the biofilm formation of UPEC cysteine auxotrophs. Here we show that cysteine auxotrophs have dysregulated biofilm components due to a change in the redox state of the periplasm. Additionally, we show the relationship between cysteine auxotrophs, biofilms, and antibiotics frequently used to treat UTIs.

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Redox analysis of the cytochrome o-type quinol oxidase complex of Escherichia coli reveals three redox components.

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