Activity of Spore-Specific Respiratory Nitrate Reductase 1 of Streptomyces coelicolor A3(2) Requires a Functional Cytochrome bcc-aa ₃ Oxidase Supercomplex

Abstract

Spores have strongly reduced metabolic activity and are produced during the complex developmental cycle of the actinobacteriumStreptomyces 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. coelicoloruses either a cytochromebdoxidase or a cytochromebcc-aa3oxidase 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 functionalbcc-aa3supercomplex to reduce nitrate. Mutants lacking the completeqcr-ctagenetic locus encoding thebcc-aa3supercomplex showed no Nar1-dependent nitrate reduction. Recovery of Nar1 activity was achieved by genetic complementation but only when the completeqcr-ctalocus 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 theqcr-ctamutant, 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 thebcc-aa3supercomplex for activity. Together, our data suggest that Nar1 functions together with the proton-translocatingbcc-aa3supercomplex to increase the efficiency of energy conservation in resting spores.IMPORTANCEStreptomyces coelicolorforms 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 cytochromebccoxidoreductase andaa3oxidase. Our findings suggest that the Nar1 enzyme in theS. coelicolorspore functions together with the proton-translocatingbcc-aa3supercomplex 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.

Anaerobic nitrate respiration in the aerobe Streptomyces coelicolor A3(2): helping maintain a proton gradient during dormancy

“…Thus, O 2 thermodynamically ‘overrides’ nitrate and consequently electrons flow to the more electro‐positive acceptor (O 2 /H 2 O, E o ’ = +815 mV; NO 3 − /NO 2 − , E o ’ = +430 mV). This idea has been borne out by a recent demonstration that Nar1 activity in spores is dependent on the presence of the O 2 ‐reducing cytochrome bcc ‐cytochrome aa 3 oxidase supercomplex (Falke et al ., ). Thus, deletion of the genes encoding the cytochrome bcc ‐cytochrome aa 3 oxidase supercomplex is sufficient to prevent manifestation of Nar1 activity, despite the enzyme being synthesized and detectable in spores of the mutant.…”

“…Thus, deletion of the genes encoding the cytochrome bcc ‐cytochrome aa 3 oxidase supercomplex is sufficient to prevent manifestation of Nar1 activity, despite the enzyme being synthesized and detectable in spores of the mutant. The dependence of Nar1 activity on the supercomplex strongly suggests that electrons are transferred directly to Nar1 from the complex, and not from quinol, and when the electron‐transfer route through the supercomplex is impeded, Nar1 becomes destabilized and inactivated (Falke et al ., ).…”

“…In the actinobacteria, complex III (cytochrome bcc oxidoreductase) and complex IV (cytochrome aa 3 oxidase) form a supercomplex through which electrons are channelled, with protein–protein interactions being critical to the process (Gong et al ., ; Wiseman et al ., ). Due to the dependence of Nar1 on the presence of the supercomplex, this suggests the intriguing possibility that Nar1 might transiently associate with this supercomplex in spores to divert electrons to the NarG1 catalytic subunit when O 2 concentrations drop below a critical threshold (< 5 μM; Falke et al ., ). At higher O 2 concentrations, it is proposed that electrons flow through the supercomplex, or ‘respirasome’ (Borrero‐de Acuña et al ., ), directly to the cytochrome aa 3 oxidase, where O 2 is reduced (Fig.…”

“…Typically, Nar enzymes act as direct quinol oxidases and use a redox‐loop mechanism to generate a proton gradient (Jormakka et al ., ). Coupling of electron transport from menaquinol (MK/MKH 2 , E o ’ = −74 mV) through the supercomplex to allow Nar1 to reduce nitrate would theoretically allow the Q‐cycle to be engaged, and thus for each 2 e − transferred, the concomitant translocation of 2 H + through the supercomplex would be possible, in addition to the 2H + protons released through menaquinol oxidation (Falke et al ., ). Such a process would enhance the efficiency of pmf generation through the use of nitrate as an acceptor when O 2 becomes limiting for spores.…”

“…Despite also being active in mycelium under hypoxic and anoxic conditions, Nar2 is not completely reliant on the bcc‐aa 3 ‐supercomplex for activity (Falke et al ., ). A further difference between the two systems is that hypoxia also induces transcription of the narG2H2J2I2 operon and this is completely dependent on the OsdKR two‐component system ( o xygen, s tress and d evelopment) in mycelium: OsdK is a membrane‐associated histidine kinase and OsdR a predicted DNA‐binding response regulator (Urem et al ., ; Fischer et al ., ).…”