Melioribacter roseus P3M-2^T was incubated at 52–54°C in the modified anaerobic S medium (Podosokorskaya et al., 2013). Sodium sulfide and resazurin were only added to a strictly anaerobic variant of the medium for fermentative growth testing (see below). In all the cases of anaerobic growth, the strain was incubated in the dark without shaking. For aerobic or microaerobic cultivation, the same medium was used, but in this case, boiling and flushing of the medium with nitrogen were omitted, and inoculated tubes or bottles were incubated with shaking. Oxygen level in aerobic and microaerobic cultures was determined at the initial point and at the end of incubation by gas chromatography as previously described (Frank Y. A. et al., 2016).

All tests were performed in the presence of 10 mM Tricine buffer and yeast extract added as a source of growth factors (0.1 g 1^-1 under anaerobic conditions and 0.05 g 1^-1 under aerobic conditions).

Two inhibitors of electron transfer—rotenone and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO)—were used for the analysis. The inhibitors (each by 15 μM) were added to the medium from anaerobic (N₂) stock solutions in 100% DMSO before cultivation. Negative control without inhibitors and additional positive controls, containing cells, acetate or maltose, air in the gas phase, and 0.1% (v/v) DMSO but no inhibitors, were used to provide data normalized to 100% activity to help estimate the impact of the inhibitors. Hydrogen was added to the gas phase (1 gauge atmosphere) to eliminate the possibility of fermentative growth of the strain on yeast extract (added to the medium as a growth factor at a concentration of 0.1 g 1^-1).

The transmembrane electrical potential (Δψ) of the bacterial cells grown under atmospheric oxygen was detected using tetraphenylphosphonium (TPP⁺) penetrating cation at the final concentration of 1.6 μM and a TPP⁺-selective electrode, as described earlier (Kamo et al., 1979) with our modifications (Muntyan et al., 2012). The experiments were conducted with washed resting cell suspensions (3 mg ml^-1 of total cell protein), prepared in fresh sterile aerobic culture medium (pH 7.0) lacking organic growth factors, in a conical thermostated glass cell at 52°C under vigorous aeration with a magnetic stirring bar. To evaluate the respiratory activity of bacterial cells grown under atmospheric oxygen, the rate of oxygen consumption was measured in the cell suspensions using a standard oxygen Clark-type electrode and a polarograph LP7e (LP, Czech Republic) at 52°C. All the measurements were performed in three biological replicas, i.e., with three different cell suspensions. Each cell suspension was divided into halves for simultaneous determination of Δψ and respiratory activity.

Phylogenetic analysis of CISM proteins was performed in the same manner as described by Sorokin et al., 2016. Phylogenetic analysis of cytochrome oxidases was performed as follows: all 725 seed sequences of Pfam PF00115 (COX1) family were downloaded from http://pfam.xfam.org/family/PF00115\#tabview=tab3. The sequences were clustered based on 50% identity threshold using CD-hit (Huang et al., 2010), and 1 representative of each cluster was left (112 sequences). Upon addition of two M. roseus homologs (MROS_0038 and MROS_1513) to this 96-sequence dataset, the sequences were aligned in MAFFT v. 7 (Katoh et al., 2002). The Le Gascuel (+I + G) model was revealed by ProtTest 2.4 (Abascal et al., 2005) to give the highest likelihood. Phylogenetic analysis was performed in MEGA v. 6 (Tamura et al., 2013).

Localization of molybdoenzymes was predicted basing on the results of six different on-line prediction services – SignalP 4.1, TatP 1.0, SecretomeP 2.0a and TMHMM 2.0 (all at CBS Prediction Servers¹), as well as PSORTb 3.0.2² and Phobius³.

For preparation of membrane fractions for proteomic analysis, M. roseus was grown fermentatively with maltose on a strictly anaerobic (E_h -130 mV vs. standard hydrogen electrode [SHE]) medium and by aerobic respiration (atmospheric oxygen) with acetate in three biological replicas, 2 liters each. Growth was controlled microscopically, and the cells were collected on the boundary of exponential and stationary growth phases by centrifugation at 14 000 g, 4°C for 15 min. Cell pellets were washed in 50 mM Tris-HCl, pH 7.5 and centrifuged at 14 000 g, 4°C for 20 min. After that, cell pellets were resuspended in 50 mM Tris-HCl, pH 7.5 with 0.1 mg ml^-1 DNAse I (Fermentas) and protease inhibitor cocktail (prepared from Protease Inhibitor Tablet, Sigma, according to manufacturer’s recommendations), and then they were sonicated on ice using Soniprep 150 Plus disintegrator (MSE, UK) for 5 min at a frequency of 12 kHz. Cell debris was separated by centrifugation at 14 000 g, 4°C for 8 min and discarded, while the supernatants were further ultracentrifuged at 100 000 g, 4°C for 1 h. Precipitated membrane fractions were stored at –80°C before analysis. Each fraction contained ca. 200 μg total cell protein as determined by the Bradford Protein Assay Kit (BioRad) and according to the manufacturer’s recommendations.

The membrane fractions were treated with 5 μl of 10% RapiGest SF (Waters) and 1 μl nuclease mix (GE Healthcare) for 30 min at 4°C, resuspended in 45 μl of 100 mM NH₄HCO₃, vortexed and heated at 100°C for 5 min. After cooling to room temperature, insoluble material was removed by centrifugation at 15 000 g for 5 min. The supernatant was separated, checked for protein concentration, subjected for disulfide bonds reduction with 10 mM 1,4-dithiothreitol (DTT, BioRad) in 100 mM ammonium bicarbonate at 60°C for 30 min, and subsequently alkylated with 30 mM iodoacetamide (BioRad) at room temperature in dark for 30 min. DTT and 100 mM ammonium bicarbonate were added iteratively. After that, alkylated trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega) was added to the supernatant in the ratio of 1/50 (mg per mg total protein) and incubated at 37°C overnight. For trypsin inactivation and degradation of the acid-labile surfactant, an aliquot of trifluoroacetic acid (Sigma) was added to the final concentration of 0.5% (m/v), and the mixture was incubated at 37°C for 45 min. Residual surfactant was removed by centrifugation at 15 000 g for 10 min. The obtained hydrolysate was desalted using a Discovery DSC-18 Tube (Supelco) according to the manufacturer’s protocol. Peptides were eluted with 700 μL 75% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA), dried in a SpeedVac (Labconco) and resuspended in 3% ACN, 0.1% TFA to the final concentration of 5 μg μl^-1.

The LC-MS/MS was performed on a TripleTOF 5600+ mass-spectrometer operating in a data-dependent mode with a NanoSpray III ion source (ABSciex, Canada) coupled to a NanoLC Ultra 2D+ nano-HPLC system (Eksigent) configured as described by Ziganshin et al. (2016).

The raw LC/MS-MS datasets (.wiff file format) were converted to Mascot Generic Format (.mgf file format) using AB SCIEX MS Data Converter (version 1.3). Proteins were identified with the Mascot search engine (version 2.5.1) against the Melioribacter roseus str. P3M sequence database (RefSeq: NC_018178, which contains 2840 amino acid sequences). The Mascot searches were performed with the following parameters: tryptic-specific peptides, maximum of one missed cleavages, a peptide charge state limited to 1+, 2+, and 3+, a peptide mass tolerance of 10 ppm, a fragment mass tolerance of 0.5 Da, and variable modifications caused by Oxidation(M) and Carbamidomethylation(C). The False Discovery Rate (FDR) was calculated using the decoy database analysis with Mascot. Individual ions score higher than 11 indicate identity or extensive homology with p < 0.05 and FDR <1%. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2014) via the PRIDE partner repository with the dataset identifier PXD003662 (refer to additional information below for details).

For comparative analysis of protein amount, emPAIs (Ishihama et al., 2005) were calculated. Data were normalized using the scaling method. Proteins were considered to be statistically different according to the unpaired two-tailed Student’s t-test (p-value <0.05) with the Benjamini and Hochberg (1995) adjustment for p-values.

For transcriptomic analysis, seven sets of primers for M. roseus genes encoding the catalytic subunits of three oxygen reductases (coxI, ccoNO, cydA) and two molybdopterin-containing oxidoreductases (ttrA and psr/phsA), as well as for the reference housekeeping genes rpoB (encoding the DNA-directed RNA polymerase subunit beta, MROS_0223) and atpA (encoding the alpha subunit of F0F1–type ATP-synthase, MROS_0272), were designed using the Primer-BLAST service (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The specificity of the primers was verified by the Sanger sequencing of amplicons. The primers are summarized in Supplementary Table S1. RT-PCR analysis was performed with the cells grown at five different cultivation conditions: aerobic respiratory growth at atmospheric O₂ concentration in the gas phase, microaerobic respiratory growth at 2% O₂ in the gas phase, anaerobic respiratory growth with arsenate, fermentative growth in the absence of external electron acceptors at positive E_h value of the medium (+200 mV vs SHE) or negative E_h (–130 mV vs SHE, corresponding to strict anaerobiosis achieved by addition of 1 mM sodium sulfide to the culture medium). At all three respiratory cultivation conditions, a non-fermentable substrate acetate was used, while at both fermentative cultivation conditions, the anaerobic media were amended with a fermentable substrate maltose. In all the cases, the total RNA was extracted using ExtractRNA and CleanRNA Standard kits followed by DNAse I treatment. To prepare cDNA, 2 μg of the total RNA was reverse-transcribed using the MMLV RT kit. All the chemicals at this stage were from Evrogen, Russia. Quantitative PCR (qPCR) was performed using the qPCRmix-HS SYBR kit (Evrogen, Russia) on a StepOnePlus^TM Real-Time PCR System (Applied Biosystems, United States). Calibration curves were constructed based on fivefold dilutions of genomic DNA of M. roseus. All growth experiments, as well as all qPCR measurements, were performed in triplicate. Transcription of the target oxidoreductase genes was normalized using the transcription level of both rpoB and atpA, which were transcribed at all the tested growth conditions in a similar ratio to each other (Supplementary Figure S1). Our final analysis was based on rpoB-normalized data.

Previous genome analysis revealed the presence of the major components of respiratory electron transfer chain (ETC) in M. roseus (Podosokorskaya et al., 2013): proton-translocating NADH-dehydrogenase complex I, membrane-bound succinate dehydrogenase/fumarate reductase, isoprenoid quinones, and quinol oxidizing alternative complex III (ACIII). To confirm the activity of this ETC in M. roseus, the influence of rotenone and HQNO on aerobic growth with maltose or acetate (a non-fermentable substrate) was evaluated. Each of the inhibitors completely arrested M. roseus growth at these conditions. Neither HQNO nor rotenone had affected fermentative growth of the organism on maltose without electron acceptors.

Rotenone is known as an inhibitor of type I NADH-dehydrogenases. This compound binds to the membranous quinone-binding subunit NuoH/Nqo8 or the interface of the subunits NuoB/D/H (Sazanov, 2012; Nichols, 2013).

2-n-heptyl-4-hydroxyquinoline-N-oxide can serve as an inhibitor of several quinone interacting enzymes. In particular, HQNO demonstrates strong inhibitory effect on complex II (Smirnova et al., 1995), as well as partial inhibition of ACIII (Refojo et al., 2010, 2012). Complete inhibition of M. roseus aerobic growth with HQNO suggests the blockage of the main electron flow leading to energy production. According to genomic analysis (Podosokorskaya et al., 2013) we can assume that the points of HQNO action could be the M. roseuscomplex II and the alternative complex III. Anyway, the observed inhibition of M. roseus aerobic growth with either rotenone or HQNO supports the presence and activity of at least two energy-transducing complexes – the complex I and, most probably, the alternative complex III.

The washed resting cells grown aerobically with acetate maintained a transmembrane electrical potential (Δψ) in the absence of added exogenous substrates, as was detected using the TPP⁺-selective electrode. The transmembrane potential dissipated upon the addition of 1 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which passively balances proton gradient across the cytoplasmic membrane (Figure 1, gray dotted line). Aliquots of these same cell preparations exhibited no respiration, as determined by a standard oxygen Clark-type electrode, unless they were supplied with exogenous energy substrates. In the presence of acetate, the cells began to respire with the oxygen consumption rate of 1.9 nmol O₂ min^-1 mg^-1 of the cell protein (kinetic data are not presented). The Δψ value on the membranes of the respiring cells was maintained at the level observed in the absence of acetate (Figure 1, black solid line). Apparently, acetate serves as an external respiratory substrate in M. roseus cells, being presumably metabolized via the acetyl-CoA synthetase-catalyzed pathway and further through the TCA cycle predicted by genome analysis (Kadnikov et al., 2013). This pathway could directly fuel the respiratory ETC via the activity of succinate dehydrogenase, resulting in initiation of oxygen consumption by the cells. The presence of the respiratory complex II in M. roseus was previously revealed by genome analysis (Kadnikov et al., 2013) and its expression under aerobic condition is supported by our proteomic data (see below, Supplementary Table S2). In the presence of the respiratory substrate (acetate) and Δψ across the bacterial cell membranes (i.e., when the respiratory chain was in partially reduced state), cyanide inhibited respiratory activity of whole resting cells. The cyanide effect on oxygen respiration was comparatively high, as 50% inhibition was observed within 10 minutes in the presence of 35 μM KCN (without preincubation) with reductive substrate, and 65% inhibition was reached at 100 μM KCN within 20 min (Supplementary Figure S2). Noteworthily, our measurements were performed under optimal growth conditions of M. roseus (pH 7.0, 52°C), at which 99% cyanide (pK = 9.2) exists in the form of easily sublimating cyanic acid (boiling point 26°C). Consequently, the active concentration of cyanide in the incubation mixture for the respiratory activity measurements was to be well below the mentioned estimated value. Additionally, the inhibitory effect of cyanide on cytochrome oxidases depends on pH of the medium, membrane environment and RedOx state of the pentacoordinated oxygen-binding heme (that is heme a₃/o₃) in the enzyme reaction center (Wilson and Erecińska, 1978; Cooper and Brown, 2008). Considering these facts and reasons, and that our measurements were performed with the whole cells and incompletely oxidized heme proteins (when the cytochrome oxidases of clade A form unstable complexes with cyanide, i.e., reversibly bind it), we assume that the actual cyanide inhibitory effect on M. roseus cytochrome oxidases is much higher, than the apparent one registered in our study. Anyway, such an effect manifests the efficiency of cyanide as an inhibitor, thus suggesting that mainly the heme-copper oxidases comprise the terminus of the respiratory chain (Nichols, 2013) in the cells grown at atmospheric oxygen concentration.

Taken together, the results of inhibitory analysis of aerobic respiration and Δψ measurements performed with M. roseus whole cells clearly highlight the activity of aerobic respiratory electron transfer chain in the microorganism. The variety and functionality of three different oxygen reductases in M. roseus are analyzed in the next section on the basis of the genomic, proteomic and transcriptomic data.

During previous preliminary genome analysis of M. roseus, three putative terminal oxidoreductases were revealed: two heme-copper cytochrome c oxidases and a quinol oxidase of the bd-type (Podosokorskaya et al., 2013). Sequence analysis of the catalytic subunits MROS_0038 and MROS_1513 (CoxI and CcoNO, respectively) and the analysis of the genomic context of their genes (Podosokorskaya et al., 2013; Karnachuk et al., 2015) revealed that CoxI is highly similar (66% amino acid sequence identity at 97% query coverage) to the atypical heme-copper cc(o/b)o₃ cytochrome oxidase with an unusual heme content, which was recently described in a strict anaerobe Desulfovibrio vulgaris Hildenborough as a proton-translocating enzyme involved in oxygen detoxification (Ramel et al., 2013). The CcoNO of M. roseus was proposed to be a typical cbb₃-type oxidoreductase with homologs in various Bacteroidetes, mainly aerobic ones (Podosokorskaya et al., 2013; Karnachuk et al., 2015). The phylogenetic analysis of coxI and ccoNO genes, performed within this work, generally reproduced the currently accepted phylogeny of heme-copper oxidoreductases (HCO; Sousa et al., 2012; Ducluzeau et al., 2014; Muntyan et al., 2015). The CoxI protein from M. roseus displayed close relation to the catalytic subunit of the cc(o/b)o₃ cytochrome oxidase described in D. vulgaris, while both enzymes appeared in the same distinct subclade of A-type oxidoreductases (Figure 2), which possess low affinity to O₂ and are proposed to be best adapted to modern atmospheric oxygen concentration (Ducluzeau et al., 2014). Analysis of protein sequences from the ‘M. roseus subclade’ (Figure 2) with a web-based HCO classifying tool (Sousa et al., 2011, www.evocell.org/hco) recognized all of them as A2-subtype oxidoreductases possessing several peculiar residues in their proton channels (Sousa et al., 2012). The CcoNO protein of M. roseus fell within the clade of the authentic cbb₃ oxidoreductases of C-type (Figure 2) notable for the mandatory Glu in the active center (Muntyan et al., 2015).

Sequence analysis of the catalytic subunit, CydA, of the bd-type quinol oxidase (MROS_0843) put this enzyme into the most widespread subfamily A of cytochromes bd with a “short Q-loop” between transmembrane helixes 6 and 7 (Borisov et al., 2011). Sequences from Bacteroidetes prevail in the first 100 best BLAST hits of MROS_0843 (UniProt database search on March 2017, Supplementary Table S3). In recent reviews, sporadic distribution of different cytochrome bd-type oxidases within the phylum Bacteroidetes was demonstrated and supposed to be a result of horizontal gene transfer. These cytochrome bd oxidases were proposed to perform various physiological functions apart from proton motive force generation, such as facilitating the colonization of O₂-poor environments or detoxifying oxygen under oxidative stress and other stressful conditions (Borisov et al., 2011; Giuffrè et al., 2014).

To investigate which of the predicted oxidases and accessory proteins are involved in oxygen respiration at atmospheric O₂ concentration, the results of a shotgun proteomic analysis of transmembrane and membrane-bound proteins were compared across two cultivation conditions: growth by aerobic respiration (at atmospheric O₂ with acetate) and maltose fermentation under strictly anaerobic conditions (at negative E_h of –130 mV vs SHE). Totally, at both growth conditions, each in three biological replicas, 1239 proteins were identified with a minimum of two unique peptides. The number of proteins identified per each sample is provided in Supplementary Tables S4–S6. For quantitative proteome analysis, exponentially modified protein abundance indexes (emPAIs) were calculated: 304 proteins were significantly different (adjusted p-value <0.05) between the cells grown by aerobic respiration and the cells grown by fermentation under strictly anaerobic conditions (Supplementary Table S2). Some proteins were identified reliably in aerobically grown cells only. Included among those proteins were (i) the catalytic subunit CoxI and the subunit CoxII of the cc(o/b)o₃ cytochrome oxidase, (ii) its redox partner class I soluble cytochrome c₅₅₁/c₅₅₂ (MROS_0033), (iii) two subunits of the alternative complex III, ActC and fused ActDE, encoded in the same cluster with the cc(o/b)o₃ oxidase by MROS_0043 and MROS_0042, respectively, and (iv) the metal ion-binding subunit of copper-transporting ATPase, MROS_1511, proposed to participate in the biogenesis of heme-copper oxidases in M. roseus (Karnachuk et al., 2015). Upregulation at aerobic versus fermentative growth was statistically significant for the following proteins (Figure 3): (i) the catalytic subunit of the ACIII complex, ActB (encoded by MROS_0044), (ii) the NADH-dehydrogenase subunit, NuoG (encoded by MROS_2032), essential to provide the catalytic site for NADH oxidation in respiratory complex I, (iii) the SCO1/SenC domain protein encoded by MROS_0039, which is located in the same cluster (MROS_0034-0039) as the cc(o/b)o₃ oxidase genes and could determine the post-translational step in the accumulation of heme-copper oxidase subunits, CoxI and CoxII (Buchwald et al., 1991), (iv) the cytochrome c biogenesis protein F, encoded by MROS_0623 (CcmF). It should be mentioned that 3-fold upregulation at aerobic conditions was also significant in the case of subunit III of the cbb₃-type oxygen reductase, while the catalytic subunit, CcoNO, of this complex was detected in only one biological replica, which does not allow for the prediction of the influence of aerobiosis on its abundance in the cells. Among proteins detected at fermentative conditions only (Supplementary Table S2) are hydrogenases of the [FeFe]-family (MROS_0634, 0635, 2480-2482, 2487, 2488), which were proposed to oxidize NADH to produce H₂ during the fermentation of sugars (Kadnikov et al., 2013), and the subunit alpha of pyruvate:ferredoxin oxidoreductase (MROS_2663), which catalyzes the final step in the Embden-Meyerhof-Parnas pathway (Kletzin and Adams, 1996). This result seems logical considering that all of the proteins suppressed under aerobic conditions are involved in the fermentative catabolism of prokaryotes. Notably, insensitivity of the cytochrome bd oxidase to the change of growth conditions supports the prediction that this enzyme does not play a role of a terminal oxygen reductase during the aerobic growth of M. roseus.

Overall, the results of the proteomic analysis, in concordance with the abovementioned biochemical data, clearly indicate the involvement of complexes I, ACIII and the atypical heme-copper cc(o/b)o₃ cytochrome oxidase in aerobic respiration in M. roseus, what is supported by the expression pattern of the proteins involved in heme-copper enzymes’ biogenesis. To our knowledge, this is the first reported evidence at protein level of the involvement of ACIII in conjunction with the cc(o/b)o₃ cytochrome oxidase in aerobic respiration. The homolog of the cc(o/b)o₃ cytochrome oxidase of M. roseus has been comprehensively characterized only in D. vulgaris, in which the involvement of this enzyme in oxygen reduction coupled to proton motive force generation was demonstrated. However, no growth stimulation by oxygen was observed for this organism, and transcriptomic data only suggested a detoxifying role of this enzyme in the metabolism of the strict anaerobe, D. vulgaris (Lamrabet et al., 2011; Ramel et al., 2013). In general, our proteomic analysis revealed induction of the cc(o/b)o₃ cytochrome oxidase (MROS_0035-0038) during aerobic growth of M. roseus in comparison to fermentative growth, while the differences in expression of the cbb₃ cytochrome oxidase proteins (MROS_1513-1515) and the bd-type oxidase (MROS_0842-0843) were not reliably detected.

Comparative transcriptomics approach was used to confirm the findings of proteomic analysis and distinguish the metabolic roles of the two different HCOs and the cytochrome bd oxidase in M. roseus. Thus, the results of the RT-PCR analysis, targeted at M. roseus genes encoding the catalytic subunits of the three oxidoreductases (coxI, ccoNO, cydA), were compared across four different cultivation conditions: respiratory aerobic growth with acetate at atmospheric O₂ concentration, microaerobic growth with acetate at 2% O₂ in the gas phase, maltose fermentation at positive E_h value of the medium (see Methods section) or at negative E_h value (i.e., at strict anaerobiosis). An anaerobic medium with positive E_h was used to assess possible involvement of the cytochrome oxidases in the oxidative stress response under the absence of oxygen in the gas phase but the presence of dissolved oxidized compounds. Strikingly, the genes of all three oxidases were transcribed at aerobic, microaerobic and both anaerobic growth conditions, although the normalized transcription level differed significantly (Figure 4). The highest normalized transcription level was observed for the coxI gene of the cc(o/b)o₃ cytochrome oxidase at aerobic conditions; the level decreased ca. 1.5-fold under microaerobic conditions (adjusted p-value 0.073, p-value 0.029) and significantly lowered 4 to 6-fold at anaerobic fermentative growth, both at negative and positive E_h (adjusted p-value ≪ 0.05 for both pairwise comparisons). Different pattern was observed for ccoNO gene of the cbb₃-type cytochrome oxidase, the transcription level of which was maximal at microaerobic cultivation conditions, did not statistically change at both aerobic or positive-E_h anaerobic fermentative conditions and approached its minimum at strict anaerobiosis, at negative E_h (adjusted p-value 0.028 for the comparison of maximal and minimal ccoNO transcription levels). Cytochrome bd oxidase demonstrated the lowest transcription levels among all three oxygen reductases at respiratory growth conditions (Figure 4). Although, transcript abundance of cydA gene was statistically indistinguishable at all tested growth conditions (adjusted p-values > 0.05 for all pairwise comparisons).

The results of transcriptomic studies demonstrate the key role of the A2-subtype cc(o/b)o₃ cytochrome oxidase as the terminal oxidoreductase in M. roseus aerobic respiration, while another heme-copper cbb₃-type oxidase is supposed to play an auxiliary role in aerobic respiration, which, however, becomes important at microaerobic conditions when a higher affinity to O₂ is needed to support the cell growth by oxygen respiration. Indeed, the oxidases of the cbb₃-type possess a high affinity to oxygen (Pitcher et al., 2002) compared to the affinity measured for a cc(o/b)o₃ cytochrome oxidase in D. vulgaris (Ramel et al., 2013). Thus, the cbb₃ oxidase of M. roseus is likely to play a major role in aerobic respiration at microaerobic conditions or, alternatively, serve for oxygen scavenging upon anaerobic fermentative growth at positive E_h (i.e., when dissolved O₂ could be present in the environment at a concentration insufficient for the activity of the cc(o/b)o₃ cytochrome oxidase). Comparatively low transcription levels of the cytochrome bd oxidase at all tested conditions allows us to suggest that this enzyme mainly serves for the scavenging or detoxification of oxygen and is not directly involved in the aerobic energy metabolism of M. roseus. Of further note, the transcription of the coxI gene was significantly higher at strict anaerobiosis than at positive-E_h fermentative conditions (adjusted p-value 0.028, Figure 4), what could reflect the similarity between the functional properties of the cc(o/b)o₃ cytochrome oxidase from M. roseus and its close homolog from the strict anaerobe, D. vulgaris, in which the detoxificating role of this oxidoreductase has been evidenced.

Interestingly, transcription of atpA (Supplementary Figure S1) and the genes of cytochrome oxidases (Figure 4) was still observed at strictly anaerobic conditions during maltose fermentation, although several enzymes involved in fermentative catabolism clearly were upregulated at these conditions, according to our proteomic analysis. The transcription of atpA during fermentation correlates with the expression data on four major F₀F₁-ATPase subunits (Supplementary Table S2) and could be explained in the view of probable functioning of the ATPase in the reverse direction of ATP hydrolysis for the dissipation of proton excess to prevent cytoplasm acidification. This function was proposed first for F₀F₁ ATPases in Clostridium pasteurianum and Thermotoga maritima pathways of acetogenic glucose fermentation, in which the key role was assigned to electron bifurcating [FeFe]-hydrogenases driving the thermodynamically unfavorable oxidation of NADH through the exergonic oxidation of ferredoxin to produce H₂ (Buckel and Thauer, 2013). In the case of M. roseus, H₂, acetate and CO₂ are the main products of maltose fermentation (Podosokorskaya et al., 2013), and one of two putative [FeFe]-hydrogenases (MROS_0634) was upregulated during maltose fermentation. Accordingly, M. roseus is likely to possess a mode of fermentative catabolism similar to that proposed for C. pasteurianum and T. maritima, involving hydrolytic activity of F₀F₁ ATPase (Buckel and Thauer, 2013). Low-level transcription of cytochrome oxidases genes during fermentative growth could be explained by the “semper paratus” state of catabolic machinery in M. roseus, considering the instability of its natural environmental conditions (Podosokorskaya et al., 2013; Frank Y. et al., 2016), that is, the low biosynthesis level of key respiratory enzymes is probably sustained by a regulome of M. roseus in order to outcompete for electron acceptors upon sharp changes to the geochemical setting.

We have previously reported on arsenate respiration in growth experiments with M. roseus (Podosokorskaya et al., 2013). Preliminary genome analysis (Podosokorskaya et al., 2013) pointed out the genes of two oxidoreductase complexes belonging to the CISM superfamily (Duval et al., 2008). Phylogenetic analysis of their molybdopterin catalytic subunits MROS_1076 and MROS_1774 (Supplementary Figure S3), performed in the frames of the current work, revealed their affiliation to tetrathionate- (Ttr) and polysulfide/thiosulfate- (Psr/Phs) reductase branches, respectively, but not to dissimilatory arsenate reductases (Arr). According to our (Supplementary Figure S3) and previous (Duval et al., 2008; Sorokin et al., 2016) reconstructions, the Ttr clade originates from the same root as the Arr clade, which, in its turn, has a common ancestor with the Psr/Phs cluster. To the moment, the only organism reported to use a non-Arr-type oxidoreductase to respire arsenate is Pyrobaculum aerophilum. This hyperthermophilic archaeon, similarly to M. roseus, possesses Ttr- and Psr/Phs-type, but not Arr-type, oxidoreductases of the CISM superfamily (Cozen et al., 2009).

Considering that no other candidates for arsenate respiration were found in M. roseus, the transcription pattern of molybdopterin catalytic subunits MROS_1076 and MROS_1774 was studied for the cells grown by respiration with acetate as the electron donor and arsenate as the sole electron acceptor under anaerobic cultivation conditions. The cells, grown with acetate under aerobic or microaerobic conditions and those grown by maltose fermentation under strict anaerobiosis (E_h –130 mV), were used as negative controls. Two sets of primers for M. roseus genes, encoding the molybdopterin catalytic subunits, were designed, and normalized transcription levels were compared across all four cultivation conditions (arsenate, aerobic, microaerobic respiration, and maltose fermentation).

The transcription of ttrA and psr/phsA genes was almost negligible in the absence of arsenate but dramatically increased in cells grown with arsenate (Figure 5). These results suggest that both ‘Psr/Phs’ and ‘Ttr’ molybdopterin oxidoreductases are involved in arsenate respiration. Yet we should note, that only the induction of psrA/phsA was clearly statistically distinguishable (adjusted p-value 0.03) in arsenate-grown cells of M. roseus. Interestingly, despite the presence of Ttr- and Psr/Phs-type enzymes as candidate arsenate reductases in both P. aerofilum and M. roseus (Supplementary Figure S3), As(V) did induce only one molybdopterin oxidoreductase in P. aerofilum – the TtrA (PAE1265, Cozen et al., 2009), while in M. roseus both the ‘Ttr’ and the ‘Psr/Phs’ oxidoreductases were induced with arsenate and the ‘Psr/Phs’ induction was more pronounced (Figure 5). Although, we cannot exclude participation of the ‘Ttr’ oxidoreductase in arsenate metabolism of M. roseus, considering occurrence of transcriptional response of the ttrA gene to arsenate and its peculiar genomic neighborhood. Encoded right upstream of the ttr locus in M. roseus genome is an ‘ArrTSR’ two-component regulatory system (Saltikov, 2011), including a phosphorylated response regulator (‘ArrR’, MROS_1070), a sensory histidine kinase (‘ArrS’, MROS_1069) and a hypothetical protein (MROS_RS05510 according to the recent NCBI RefSeq reannotation), homologous to a periplasmic phosphonate binding protein ‘ArrT’ (Saltikov, 2011), a part of phosphonate ABC transporters (Alicea et al., 2011). The ‘ArrTSR’ system is supposed to induce transcription of dissimilatory arsenate reductases in response to periplasmic arsenate via the ‘ArrR’ regulator (Saltikov, 2011). Finally, in M. roseus in close vicinity to arrT gene is an ars-type arsenic resistance locus arsRPCB (MROS_1063-1067, Kadnikov et al., 2013), encoding detoxificating glutathione:arsenate oxidoreductase ArsC, an arsenite exporter ArsB and a specific regulator ArsR, which induces transcription of arsPCB in the presence of arsenite (Saltikov, 2011). In contrast to ttr, the psr/phs locus is preceded by the gene of MerR-like transcription activator (MROS_1773), which can respond to heavy metal ions and chemical stresses (Brown et al., 2003). Peculiarities of genomic environment of ttrA and psrA/phsA genes allow us to hypothesize that ‘PsrA/PhsA’ and ‘TtrA’ enzymes play different roles in arsenate respiration of M. roseus. We propose the ‘PsrA/PhsA’ to be the major arsenate reductase, as its transcription could be directly induced by As(V) and such an induction is supported by our experimental data. On the other hand, ‘TtrA’ could act as an auxiliary arsenate reductase which activity could be co-regulated with arsenic detoxification complex ArsPCB. Thus, our results indicate utilization of two non-Arr arsenate reductases by M. roseus in arsenic respiratory metabolism (Figure 6), yet further studies are definitely needed to cast light on their exact physiological functions.