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Mitochondrial alternative oxidase (AOX) is predicted to be present in mitochondria of several invertebrate taxa including tardigrades. Independently of the reason concerning the enzyme occurrence in animal mitochondria, expression of AOX in human mitochondria is regarded as a potential therapeutic strategy. Till now, relevant data were obtained due to heterologous AOX expression in cells and animals without natively expressed AOX. Application of animals natively expressing AOX could importantly contribute to the research. Thus, we decided to investigate AOX activity in intact specimens of the tardigrade Hypsibius exemplaris. We observed that H. exemplaris specimens’ tolerance to the blockage of the mitochondrial respiratory chain (MRC) cytochrome pathway was diminished in the presence of AOX inhibitor and the inhibitor-sensitive respiration enabled the tardigrade respiration under condition of the blockage. Importantly, these observations correlated with relevant changes of the mitochondrial inner membrane potential (Δψ) detected in intact animals. Moreover, detection of AOX at protein level required the MRC cytochrome pathway blockage. Overall, we demonstrated that AOX activity in tardigrades can be monitored by the animals’ behavior observation as well as by measurement of intact specimens’ whole-body respiration and Δψ. Furthermore, it is also possible to check the impact of the MRC cytochrome pathway blockage on AOX level as well as AOX inhibition in the absence of the blockage on animal functioning. Thus, H. exemplaris could be consider as a whole-animal model suitable to study AOX.

Small proteins are a new and expanding area of research. Many characterized small proteins are composed of a single hydrophobic α-helix, and the functional requirements of their limited amino acid sequence are not well understood. One hydrophobic small protein, CydX, has been shown to be a component of the cytochrome bd oxidase complex in Escherichia coli, and is required for enzyme function. To investigate small protein sequence specificity, an alanine scanning mutagenesis on the small protein CydX was conducted using mutant alleles expressed from the E. coli chromosome at the wild-type locus. The resulting mutant strains were assayed for CydX function. No single amino acid was required to maintain wild-type resistance to β-mercaptoethanol. However, substitutions of 10-amino acid blocks indicated that the N-terminus of the protein was required for wild-type CydX activity. A series of double mutants showed that multiple mutations at the N-terminus led to β-mercaptoethanol sensitivity in vivo. Triple mutants showed both in vivo and in vitro phenotypes. Together, these data provide evidence suggesting a high level of functional plasticity in CydX, in which multiple amino acids may work cooperatively to facilitate CydX function.

Chromosome replication in Escherichia coli is initiated by DnaA. DnaA binds ATP which is essential for formation of a DnaA-oriC nucleoprotein complex that promotes strand opening, helicase loading and replisome assembly. Following initiation, DnaAATP is converted to DnaAADP primarily by the Regulatory Inactivation of DnaA process (RIDA). In RIDA deficient cells, DnaAATP accumulates leading to uncontrolled initiation of replication and cell death by accumulation of DNA strand breaks. Mutations that suppress RIDA deficiency either dampen overinitiation or permit growth despite overinitiation. We characterize mutations of the last group that have in common that distinct metabolic routes are rewired resulting in the redirection of electron flow towards the cytochrome bd-1. We propose a model where cytochrome bd-1 lowers the formation of reactive oxygen species and hence oxidative damage to the DNA in general. This increases the processivity of replication forks generated by overinitiation to a level that sustains viability., Author Summary In most bacteria chromosome replication is initiated by the DnaA protein. In Escherichia coli, DnaA binds ATP and ADP with similar affinity but only the ATP bound form is active. An increased level of DnaAATP causes overinitiation and cell death by accumulation of DNA strand breaks. These strand breaks often result from forks encountering gapped DNA formed during repair of oxidative damage. We provide evidence that cell death in overinitiating cells can be prevented by rewiring the metabolism to favor the micro-aerobic respiratory chain with the cytochrome bd-1 as terminal oxidase. Cytochrome bd-1 is found in aerobic as well as anaerobic bacteria. Its role is to reduce O2 in micro-aerobic conditions and work as an electron sink to prevent the formation of reactive oxygen species. Our results suggest that bacteria can cope with replication stress by increasing respiration through cytochrome bd-1 to reduce the formation of reactive oxygen species, and hence oxidative damage to a level that does not interfere with replication fork progression.

For decades respiratory chain and photosystems were the main firing field of the studies devoted to mechanisms of electron transfer in proteins. The concept of conjugated lateral electron and transverse proton transport during cellular respiration and photosynthesis, which was formulated in the beginning of 1960-s, has been confirmed by thousands of experiments. However, charge transfer in recently discovered bacterial nanofilaments produced by various electrogenic bacteria is regarded currently outside of electron and proton conjugation concept. Here we report the new study of charge transfer within nanofilaments produced by Shewanella oneidensis MR-1 conducted in atmosphere of different relative humidity (RH). We utilize impedance spectroscopy and DC (direct current) transport measurements to find out the peculiarities of conductivity and Raman spectroscopy to analyze the nanofilaments' composition. Data analysis demonstrates that apparent conductivity of nanofilaments has crucial sensitivity to humidity and contains several components including one with unusual behavior which we assign to electron transport. We demonstrate that in the case of Shewanella oneidensis MR-1 charge transfer within these objects is strongly mediated by water. Basing on current data analysis of conductivity we conclude that the studied filaments of Shewanella oneidensis MR-1 are capable of hybrid (conjugated) electron and ion conductivity.