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Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa₃ oxidase supercomplex from spores of Streptomyces coelicolor A3(2).

ABSTRACT: In order to reduce nitrate in vivo, the spore-specific respiratory nitrate reductase, Nar1, of Streptomyces coelicolor relies on an active cytochrome bcc-aa₃ oxidase supercomplex (bcc-aa₃ supercomplex). This suggests that membrane-associated Nar1, comprising NarG1, NarH1, and NarI1 subunits, might not act as a classical menaquinol oxidase but could either receive electrons from the bcc-aa₃ supercomplex, or require the supercomplex to stabilize the reductase in the membrane to allow it to function. To address the biochemical basis for this dependence on the bcc-aa₃ supercomplex, we purified two different Strep-tagged variants of Nar1 and enriched the native enzyme complex from spore extracts using different chromatographic and electrophoretic procedures. Polypeptides associated with the isolated Nar1 complexes were identified using mass spectrometry and included components of the bcc-aa₃ supercomplex, along with an alternative, spore-specific cytochrome b component, QcrB3. Surprisingly, we also co-enriched the Nar3 enzyme with Nar1 from the wild-type strain of S. coelicolor. Two differentially migrating active Nar1 complexes could be identified after clear native polyacrylamide gel electrophoresis; these had masses of approximately 450 and 250 kDa. The distribution of active Nar1 in these complexes was influenced by the presence of cytochrome bd oxidase and by QcrB3; the presence of the latter shifted Nar1 into the larger complex. Together, these data suggest that several respiratory complexes can associate in the spore membrane, including Nar1, Nar3, and the bcc-aa₃ supercomplex. Moreover, these findings provide initial support for the hypothesis that Nar1 and the bcc-aa₃ supercomplex physically associate.

SUBMITTER: Falke D

PROVIDER: S-EPMC7931247 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Spores have strongly reduced metabolic activity and are produced during the complex developmental cycle of the actinobacterium Streptomyces coelicolor Resting spores can remain viable for decades, yet little is known about how they conserve energy. It is known, however, that they can reduce either oxygen or nitrate using endogenous electron sources. S. coelicolor uses either a cytochrome bd oxidase or a cytochrome bcc-aa 3 oxidase supercomplex to reduce oxygen, while nitrate is reduced by Nar-type nitrate reductases, which typically oxidize quinol directly. Here, we show that in resting spores the Nar1 nitrate reductase requires a functional bcc-aa 3 supercomplex to reduce nitrate. Mutants lacking the complete qcr-cta genetic locus encoding the bcc-aa 3 supercomplex showed no Nar1-dependent nitrate reduction. Recovery of Nar1 activity was achieved by genetic complementation but only when the complete qcr-cta locus was reintroduced to the mutant strain. We could exclude that the dependence on the supercomplex for nitrate reduction was via regulation of nitrate transport. Moreover, the catalytic subunit, NarG1, of Nar1 was synthesized in the qcr-cta mutant, ruling out transcriptional control. Constitutive synthesis of Nar1 in mycelium revealed that the enzyme was poorly active in this compartment, suggesting that the Nar1 enzyme cannot act as a typical quinol oxidase. Notably, nitrate reduction by the Nar2 enzyme, which is active in growing mycelium, was not wholly dependent on the bcc-aa 3 supercomplex for activity. Together, our data suggest that Nar1 functions together with the proton-translocating bcc-aa 3 supercomplex to increase the efficiency of energy conservation in resting spores.IMPORTANCE Streptomyces coelicolor forms spores that respire with either oxygen or nitrate, using only endogenous electron donors. This helps maintain a membrane potential and, thus, viability. Respiratory nitrate reductase (Nar) usually receives electrons directly from reduced quinone species; however, we show that nitrate respiration in spores requires a respiratory supercomplex comprising cytochrome bcc oxidoreductase and aa 3 oxidase. Our findings suggest that the Nar1 enzyme in the S. coelicolor spore functions together with the proton-translocating bcc-aa 3 supercomplex to help maintain the membrane potential more efficiently. Dissecting the mechanisms underlying this survival strategy is important for our general understanding of bacterial persistence during infection processes and of how bacteria might deal with nutrient limitation in the natural environment.

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Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa₃ oxidase supercomplex from spores of Streptomyces coelicolor A3(2).

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Dataset Information

Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa3 oxidase supercomplex from spores of Streptomyces coelicolor A3(2).

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa₃ oxidase supercomplex from spores of Streptomyces coelicolor A3(2).