Marine Schoeffler

Anne-Laure Gaudin

Fanny Ramel

Odile Valette

Yann Denis

Wagdi Ben Hania

Agnès Hirschler-Réa

Alain Dolla
email: alain.dolla@mio.osupytheas.fr

Growth of an anaerobic sulfate-reducing bacterium sustained by oxygen respiratory energy conservation after O 2 -driven experimental evolution

Desulfovibrio species are representatives of microorganisms at the boundary between anaerobic and aerobic lifestyles, since they contain the enzymatic systems required for both sulfate and oxygen reduction. However, the latter has been shown to be solely a protective mechanism. By implementing the oxygen-driven experimental evolution of Desulfovibrio vulgaris Hildenborough, we have obtained strains that have evolved to grow with energy derived from oxidative phosphorylation linked to oxygen reduction. We show that a few mutations are sufficient for the emergence of this phenotype and reveal two routes of evolution primarily involving either inactivation or overexpression of the gene encoding heterodisulfide reductase. We propose that the oxygen respiration for energy conservation that sustains the growth of the O 2 -evolved strains is associated with a rearrangement of metabolite fluxes, especially NAD + /NADH, leading to an optimized O 2 reduction. These evolved strains are the first sulfate-reducing bacteria that exhibit a demonstrated oxygen respiratory process that enables growth.

Introduction

Sulfate-reducing bacteria (SRB) are ubiquitously distributed anaerobic microorganisms that gain energy for growth by coupling the oxidation of organic compounds or molecular hydrogen with the reduction of sulfate to sulfide [START_REF] Thauer | Energy metabolism and phylogenetic diversity of sulphatereducing bacteria[END_REF]. Despite the anaerobic nature of this metabolic process, SRB activity is not confined to permanently anoxic habitats since it has also frequently been found in biotopes that are temporarily exposed to oxygen [START_REF] Canfield | Aerobic sulfate reduction in microbial mats[END_REF][START_REF] Minz | Unexpected population distribution in a microbial mat community: sulfate-reducing bacteria localized to the highly oxic chemocline in contrast to a eukaryotic preference for anoxia[END_REF][START_REF] Fourçans | Molecular analysis of the spatio-temporal distribution of sulfate-reducing bacteria (SRB) in Camargue (France) hypersaline microbial mat[END_REF]. Several types of ecological data, as well as impact studies of temporary oxygen stresses on various SRB [START_REF] Marschall | Influence of oxygen on sulfate reduction and growth of sulfatereducing bacteria[END_REF][START_REF] Risatti | Community structure of a microbial mat: the phylogenetic dimension[END_REF][START_REF] Krekeler | Strategies of sulfate-reducing bacteria to escape oxygen stress in a cyanobacterial mat[END_REF][START_REF] Fournier | Response of the anaerobe Desulfovibrio vulgaris Hildenborough to oxidative conditions: proteome and transcript analysis[END_REF][START_REF] Mukhopadhyay | Cell-wide responses to low-oxygen exposure in Desulfovibrio vulgaris Hildenborough[END_REF][START_REF] Vita | Disulfide bond-dependent mechanism of protection against oxidative stress in pyruvate-ferredoxin oxidoreductase of anaerobic Desulfovibrio bacteria[END_REF][START_REF] Figueiredo | Bacterioferritin protects the anaerobe Desulfovibrio vulgaris Hildenborough against oxygen[END_REF][START_REF] Ramel | Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation[END_REF] demonstrated the aerotolerance capabilities of these bacteria with a higher oxygen tolerance for the members of the genus Desulfovibrio [START_REF] Cypionka | Survival of sulfate-reducing bacteria after oxygen stress, and growth in sulfate-free oxygen sulfide gradients[END_REF].

Desulfovibrio species have developed strategies to address the presence of oxygen and to protect themselves against its harmful effects, such as by the use of reactive oxygen species scavenging and oxygen consumption [START_REF] Dolla | Oxygen defense in sulfate-reducing bacteria[END_REF]. Oxygen consumption by Desulfovibrio species occurs in the three cell compartments. In the periplasm, this activity involves hydrogenases and c-type cytochromes [START_REF] Baumgarten | Periplasmic oxygen reduction by Desulfovibrio species[END_REF][START_REF] Fournier | A new function of the Desulfovibrio vulgaris Hildenborough [Fe] hydrogenase in the protection against oxidative stress[END_REF]. In the cytoplasm, rubredoxinoxygen oxidoreductase (ROO) is the terminal enzyme of a cytoplasmic NADH-linked chain that reduces oxygen in water [START_REF] Chen | Rubredoxin oxidase, a new flavo-hemoprotein, is the site of oxygen reduction to water by the "strict anaerobe" Desulfovibrio gigas[END_REF][START_REF] Frazão | Structure of a dioxygen reduction enzyme from Desulfovibrio gigas[END_REF]. Finally, in the membrane, this activity is performed by two oxygen reductases, a cytochrome c oxidase and a bd quinol oxidase, homologues of the enzymes found in aerobic microorganisms [START_REF] Lemos | The 'strict' anaerobe Desulfovibrio gigas contains a membrane-bound oxygen-reducing respiratory chain[END_REF][START_REF] Santana | Presence and expression of terminal oxygen reductases in strictly anaerobic sulfate-reducing bacteria isolated from salt-marsh sediments[END_REF][START_REF] Lamrabet | Environmental Microbiology O 2 -driven experimental evolution of Desulfovibrio 11 reduction in the strict anaerobe Desulfovibrio vulgaris Hildenborough: characterization of two membrane-bound oxygen reductases[END_REF]. A striking feature of these microorganisms is that they contain enzymes required for both sulfate and oxygen reduction [START_REF] Lamrabet | Environmental Microbiology O 2 -driven experimental evolution of Desulfovibrio 11 reduction in the strict anaerobe Desulfovibrio vulgaris Hildenborough: characterization of two membrane-bound oxygen reductases[END_REF][START_REF] Pereira | A comparative genomic analysis of energy metabolism in sulfate reducing bacteria and archaea[END_REF]. However, sulfate reduction is a true respiratory process that yields energy through oxidative phosphorylation to sustain growth [START_REF] Thauer | Energy metabolism and phylogenetic diversity of sulphatereducing bacteria[END_REF][START_REF] Keller | Genetics and molecular biology of the electron flow for sulfate respiration in Desulfovibrio[END_REF], while no growth dependent upon energy derived from oxygen reduction has ever been clearly demonstrated in pure Desulfovibrio cultures. Thus, oxygen reduction is thought to be a protective mechanism related to the high aerotolerance of these bacteria [START_REF] Mukhopadhyay | Cell-wide responses to low-oxygen exposure in Desulfovibrio vulgaris Hildenborough[END_REF][START_REF] Ramel | Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation[END_REF]2015).

Because of their enzymatic repertoire, Desulfovibrio species are good representatives of microorganisms at the boundary between anaerobic and aerobic respirations. In this study, we used the O 2 -driven experimental evolution of Desulfovibrio vulgaris Hildenborough (DvH) to obtain additional insight into the ways by which this anaerobic microorganism copes with the presence of oxygen. We obtained evolved strains that could not only grow in the presence of a 32-fold higher O 2 concentration than the maximal concentration tolerated by the ancestral DvH strain, but also utilize a true oxygen respiratory process to conserve energy to sustain growth in the total absence of sulfate. Two distinct routes of evolution to acquire this phenotype were observed with only a few mutations sufficient to permit the emergence of this new metabolic capability. We propose that metabolic flux rearrangement in the O 2 -evolved strains is the key mechanism that ultimately enables the optimization of O 2 respiration to sustain growth.

Results

O 2 -driven experimental evolution

To investigate the adaptation strategy of DvH to oxidative conditions, a culture of a clonal isolate of the ancestral DvH strain (WTOR) was successively subcultured in lactate/sulfate medium under continuous oxygen sparging with O 2 concentrations increasing from 0.02% to 0.65% (i.e., 0.23 μM to 7.7 μM dissolved O 2 ) for a total of 114 generations. As a control, an aliquot of the WTOR culture was successively subcultured in lactate/sulfate medium under anaerobic conditions (Supporting Information Fig. S1) for the same number of generations. At the end, we obtained a population of O 2 -evolved cells able to grow to a final OD 600nm of ~1 under continuous 0.65% O 2 -sparging while the control, a population of anaerobicevolved cells obtained after 114 generations, was unable to grow under this condition (Fig. 1A). Thus, O 2 -driven adaptive laboratory evolution permitted the selection of a DvH population exhibiting a greater aerotolerance than the WTOR strain. It should be noted that attempts to further increase the oxygen concentration in the gas mix for sparging were unsuccessful, suggesting that this 7.7 μM of dissolved O 2 is the maximum concentration tolerated by the evolved DvH population under these growth conditions.

Twenty-five clones from the O 2 -evolved population (ES strains) were randomly selected after plating under anaerobic conditions and re-grown in liquid lactate/sulfate medium under continuous 0.65% O 2 -sparging conditions. At the same time, five clones from the anaerobic-evolved population (114G strains) were also randomly selected after plating under anaerobic conditions and re-grown in liquid lactate/sulfate medium under anaerobic conditions. All 25 ES strains grew to a final OD 600nm of ~1 under continuous 0.65% O 2 -sparging conditions while none of the five 114G strains nor the WTOR did (data not shown).

Genotypic characterization

As a first step, the expression of genes encoding known oxygen reductase enzymes in DvH [START_REF] Dolla | Oxygen defense in sulfate-reducing bacteria[END_REF] in the isolated strains was analysed by qPCR. Surprisingly, some of the strains lacked an amplification signal corresponding to the roo1 gene (DVU2014) encoding a ROO. The presence of the roo1 gene was confirmed in only 6 of the 25 ES strains (data not shown). The roo1 gene is located in a genomic island (GEI) and it has been shown that the GEI can be excised and lost during successive cultivation of DvH [START_REF] Johnston | A genomic Island of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough promotes survival under stress conditions while decreasing the efficiency of anaerobic growth[END_REF]. Absence of the roo1 gene can be thus related to the absence of the GEI in these ES strains. Thus, 24% of the ES strains isolated (6 out of 25) contained the genomic island GEI, while 76% (19 out of 25) lacked it. This showed that the O 2 -evolved population obtained at the end of the O 2 -driven adaptive laboratory evolution was composed of a mixture of GEI + and GEI -cells. To assess the distribution of these two genotypes during the course of the O 2 -driven adaptive laboratory evolution, the number of copies of the roo1 gene, located within the GEI, was quantified by qPCR and related to that of the roo2 gene (DVU3185), located outside the GEI.

The number of copies of roo1 relative to the number of copies of roo2 in the WTOR culture was close to 1 (Fig. 2), showing that the ancestral clonal DvH strain was GEI + . However, this number drastically decreased during the first steps of the O 2 -driven adaptive evolution to only approximately 0.2 after 44 generations in the culture exposed to continuous 0.12% O 2 -sparging conditions. In contrast, this ratio was approximately one in the anaerobic-evolved culture after 114 generations (Fig. 2). Thus, the decrease in frequency of the GEI in the O 2 -evolved population was triggered by the presence of oxygen and occurred early along the course of the O 2 -driven adaptive laboratory evolution.

The genomes of six ES strains, three containing the GEI, namely, ESC, ESD and ESE, as well as three that lacked it, namely, ES3, ES4 and ES5, were sequenced using Illumina technology. The genomes of three 114G strains (114G1, 114G2 and 114G3) and of the ancestral DvH strain (WTOR) were also sequenced (Supporting Information Table S1). Comparative analysis with the NCBI reference DvH sequence NC_002937.3 [START_REF] Heidelberg | The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough[END_REF] led to the identification of several single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) mutations.

Mutations found in WTOR genome compared with the NCBI reference DvH sequence NC_002937.3 (Supporting Information Table S2) were shared by the ES and 114G strains. They were excluded from the analysis, since they represented mutations resulting from the propagation of the DvH strain in the laboratory for years. In contrast, mutations exclusively found in the ES and 114G strains were considered to be the result of O 2driven and anaerobic adaptive laboratory evolutions respectively. Among them, eight polymorphic loci in the WTOR at low frequencies (below 0.05 from NGS data) but homogenous alleles in the isolated ES or 114G strains were identified (Table 1). This suggested that these pre-existing mutations were under selection during the laboratory evolution experiment. Considering the mutations shared by all the ES strains but not present in the 114G strains, two distinct genotypes were revealed and thus could account for the observed phenotype of growth under continuous 0.65% O 2 -sparging. One genotype, corresponding to strains ES3, 4 and 5, was characterized by the absence of the GEI, a point mutation in the hdrA gene (DVU2402) inducing a frameshift in the coding region and an insertion upstream from the transcriptional start of DVU1690, which encodes a transcriptional regulator, TetR. Further analysis by PCR genotyping showed that these mutations were present in all the plate-isolated strains that did not contain the GEI. The second genotype, corresponding to strains ESC, D and E, was characterized by the presence of the GEI and the deletion of four nucleotides upstream of DVU1690. This deletion was confirmed in all the plate-isolated ES strains that contained the GEI. It should be noted that the mutation in the hdrA gene (DVU2402) was from the pre-existing polymorphisms in the ancestral DvH strain while both mutations upstream from the DVU1690 gene were new mutations that appeared early during the course of the O 2 -driven evolution (detectable in the culture under 0.04% O 2 sparging) (Supporting Information Fig. S2).

The O 2 -driven experimental evolution allowed us to obtain evolved strains of DvH with greater aerotolerance than the WTOR strain, after the limited number of 114 generations and only a limited number of genomic mutations. These strains can grow under continuous 0.65% oxygen-sparging corresponding to 7.7 μM of dissolved oxygen at 33 C, approximately 32-fold higher than the maximal concentration tolerated by the ancestral DvH strain [START_REF] Ramel | Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases[END_REF]. Phenotypes of the ES5 2.

The final biomass of the O 2 -evolved strains grown with 0.65% O 2 , measured as the final OD 600nm , and growth rates in lactate/sulfate medium, were as high as those of the ancestral WTOR and 114G2 strains grown under anaerobic conditions (Table 2, Supporting Information Fig. S3). It should be noted that cell counting by microscopy revealed a similar number of cells per mL for a given OD 600nm (1 unit at OD 600nm corresponding to 7.21 AE 1.13 10 8 cell mL -1 ) regardless of the strain or the growth conditions (i.e., O 2 -sparging or anaerobic conditions). As expected, at the end of growth, the ESE strain cultured with O 2 consumed almost all the lactate, similar to the WTOR and 114G2 strains cultured under anaerobiosis. In the case of the ES5 strain, approximately 15% of the lactate remained (Supporting Information Fig. S5A). The molar growth yield on lactate (Y lac ) was slightly higher for the ES5 and ESE strains than for WTOR (Supporting Information Fig. S5B). The metabolism of the O 2 -evolved strains when grown with O 2 appeared to be as efficient as that of the ancestral WTOR strain grown under anaerobic conditions, indicating that the O 2 -evolved strains were well-adapted to the oxidative conditions. Interestingly, the lactate consumed -to-sulfate reduced ratio was higher for the O 2evolved strains grown with O 2 than for the WTOR and 114G2 strains under anaerobic conditions (Supporting Information Fig. S5C), suggesting that the O 2 -evolved strains used O 2 as an alternative electron acceptor in addition to sulfate.

To confirm this hypothesis, the cells were grown in lactate/sulfate-limiting medium (37 mM lactate/9 mM sulfate) (Supporting Information Fig. S3). As expected, the final biomass for all the type strains cultured under anaerobiosis was lower than that reached when sulfate was not limiting. Interestingly, both the biomass and growth rate of the ES strains were significantly higher when cultured with O 2 than under anaerobic conditions (Table 2). At the end of the growth, sulfate was completely consumed in all the cultures (data not shown). Under oxygen sparging conditions, sulfate was consumed throughout the duration of the growth, explaining the lack of diauxic growth kinetics (Supporting Information Fig. S6). It should be noted that thiosulfate or sulfite could not be detected by ion chromatography. When 250 μM molybdate, an inhibitor of sulfate reduction [START_REF] Newport | The mechanisms of inhibition of Desulfovibrio and Desulfotomaculum species by selenate and molybdate[END_REF], was added to the medium, the final biomass when both the ESE and ES5 strains were cultured under continuous 0.65% oxygensparging was quite similar, while it was drastically reduced when the strains were cultured under anaerobic conditions, corresponding to only 48% and 51% of the final biomass in the absence of molybdate for ESE and ES5 respectively (Supporting Information Fig. S7). It has been observed that some Desulfovibrio strains developed atypically elongated cells when grown in the presence of oxygen [START_REF] Sass | Psychrotolerant sulfatereducing bacteria from an oxic freshwater sediment, description of Desulfovibrio cuneatus sp. nov. and Desulfovibrio litoralis sp[END_REF] and in a recent study, we showed that when DvH was cultured in lactate/ sulfate medium under continuous 0.02% O 2 sparging, the median length of the cells increased by 0.23 μm [START_REF] Ramel | Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases[END_REF]. Analysis of the cell length by TEM showed that, when cultured under anaerobic conditions, the length distribution of the WTOR and the ES cells were comparable with a median value of 1.27 μm, 1.43 μm and 1.20 μm for the WTOR, ES5 and ESE cells, respectively, while the median length of the 114G2 cells was higher (2.01 μm). When grown under continuous O 2 -sparging, the median cell lengths of the ES5 and ESE cells shifted to larger values, +0.39 μm and + 0.36 μm respectively (Supporting Information Fig. S8). As for DvH cultured under constant low oxygen sparging (0.02% O 2 ) [START_REF] Ramel | Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases[END_REF], culturing the O 2 -evolved strains under continuous 0.65% O 2 sparging induces comparable morphological changes that are reversible when the cells are grown under anaerobic conditions.

Growth of the ES strains with O 2 as the terminal electron acceptor

Cultivation of the ES strains was attempted with lactate as carbon and energy sources and oxygen as the electron acceptor in sulfate-free medium. In this medium, sulfur source is provided by the yeast extract. However, all the assays were unsuccessful, regardless of the addition of strong reducer such as cysteine-HCl, Na 2 S or titanium citrate. Since the standard free energy of pyruvate dehydrogenation is approximately 11-fold lower than that of lactate [START_REF] Thauer | Energy conservation in chemotrophic anaerobic bacteria[END_REF], growth assays in sulfatefree medium were performed using pyruvate instead of lactate as electron donors. Remarkably, the growth of both ES5 and ESE strains in the total absence of sulfate under continuous 0.65% O 2 -sparging were observed with a maximal OD 600nm obtained after approximately 40 h at 33 C, while neither WTOR nor the 114G2 strain grew during the same time period (Fig. 1B and Table 2). In the absence of both oxygen and sulfate, no growth was observed by any strain (ES, 114G or WTOR) during the same time period (Table 2, Supporting Information Fig. S4). Because during pyruvate fermentation, the produced H 2 could inhibit the growth, cultures were attempted under continuous N 2 -sparging (40 ml min -1 ) to lower the H 2 partial pressure in the medium. However, none of the strains grew during the same time period (40 h). It should be noted that the final biomass when the ESE and ES5 strains were cultured in pyruvate/sulfatefree medium under continuous 0.65% O 2 -sparging was similar without and with addition of 250 μM molybdate. As molybdate inhibition control, under anaerobic conditions in pyruvate/sulfate medium, the final biomasses obtained with ESE and ES5 were much lower in the presence of 250 μM molybdate than it is absence (64% and 71% lower respectively) (Supporting Information Fig. S7). Because the strains were stored frozen in lactate/sulfate, it is conceivable that some hydrogen sulfide could be added to the medium with the inoculum even if two successive subcultures were preliminary performed in pyruvate/sulfate-free medium. Hydrogen sulfide concentrations in the ES5 and ESE cultures just after inoculation were 0.01 AE 0.01 mM and 0.03 AE 0.02 mM respectively. Thus, this very low H 2 S concentration at the inoculation time could not account for the observed growth of the ES strains in pyruvate/sulfate-free medium under continuous oxygen sparging via the biological or abiotic sulfide oxidation that would occur [START_REF] Fuseler | A common pathway of sulfide oxidation by sulfatereducing bacteria[END_REF][START_REF] Luther | Thermodynamics and kinetics of sulfide oxidation by oxygen: a look at inorganically controlled reactions and biologically mediated processes in the environment[END_REF]. End-product analysis by HPLC of pyruvate metabolism revealed that acetate was the only organic acid detected. Neither fumarate, formate, succinate nor ethanol, proposed as possible end-products of pyruvate fermentation in addition to acetate in D. vulgaris Hildenborough [START_REF] Voordouw | Carbon monoxide cycling by Desulfovibrio vulgaris Hildenborough[END_REF][START_REF] Ramos | The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris Hildenborough[END_REF] or D. alaskensis G20 [START_REF] Meyer | The energy-conserving electron transfer system used by Desulfovibrio alaskensis strain G20 during pyruvate fermentation involves reduction of endogenously formed fumarate and cytoplasmic and membrane-bound complexes, Hdr-Flox and Rnf[END_REF] could be detected under any growth conditions (i.e., in the presence of sulfate under anaerobic conditions or in its absence under continuous O 2 -sparging). The addition of 6 mM KCN, an inhibitor of the membrane-bound oxygen reductases [START_REF] Ramel | Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation[END_REF], in the culture medium completely inhibited the growth of both strains in pyruvate/sulfatefree medium under continuous 0.65% O 2 -sparging while the final biomass was similar for both strains in pyruvate/sulfate medium under anaerobic conditions with or without the addition of KCN (Supporting Information Fig. S9).

The energy derived from pyruvate oxidation, measured as the intracellular ATP concentration in mid-log phase cells, was approximately three times higher when the ES strains were cultured with O 2 in the absence of sulfate than when they were cultured anaerobically in the presence of sulfate (Fig. 3). The expected free energy change of the oxygen-dependent pyruvate oxidation (Eq. 1) (ΔG '0 = -284.5 kJ mol -1 of pyruvate) is 3.3-fold more exergonic than that of the sulfate-dependent pyruvate oxidation (Eq. 2) (ΔG '0 = -85.3 kJ mol -1 of pyruvate) [START_REF] Thauer | Energy conservation in chemotrophic anaerobic bacteria[END_REF].

CH 3 COCOOH + 0:5 O 2 + H 2 O ! CH 3 COOH + HCO 3 -+ H + ð1Þ
CH 3 COCOOH + 0:25 SO 4 2-+ H 2 O ! CH 3 COOH + HCO 3 -+ 0:75 H + + 0:25 HS - ð2Þ
Considering the energy required to synthesize ATP from ADP and inorganic phosphate [approximately 75 kJ mol -1 [START_REF] Thauer | Energy conservation in chemotrophic anaerobic bacteria[END_REF]], more ATP could be synthesized when using O 2 as an electron acceptor instead of sulfate. These theoretical values are in complete agreement with the intracellular ATP concentrations experimentally measured that are greater in the ES strains cultured with O 2 .

These phenotypic analyses of the O 2 -evolved strains in sulfate-free medium unambiguously demonstrate that they respire oxygen as a true respiratory process and gain sufficient energy through oxidative phosphorylation to sustain growth.

Mechanism of evolution

To obtain deeper insights into the manner by which the O 2 -evolved strains were able to grow by respiring oxygen, the expression level of the genes encoding bd quinol oxidase, cytochrome c oxidase and ROO was analysed by qPCR. It revealed that only the transcription of the bd genes was increased in both the ES5 and ESE strains [4.4 AE 0.5 and 6.3 AE 0.6 times (mean AE SD, n = 9) respectively] compared with the WTOR strain. The oxygen consumption activities of the cells were measured. The overall oxygen consumption activities in the washed whole cells with lactate as an electron donor were 25.14 AE 4.59, 22.36 AE 4.50 and 25.50 AE 3.55 nmol O 2 consumed/min/mg protein for the WTOR, ES5 and ESE strains respectively. Despite the bd gene upregulation, no significant increase in the O 2 consumption activity was measured in the ES strains compared with the WTOR strain. It had been proposed previously that the accumulation of internal carbon reserves can be used as reducing power to reduce oxygen in Desulfovibrio gigas [START_REF] Santos | Aerobic metabolism of carbon reserves by the "obligate anaerobe" Desulfovibrio gigas[END_REF]. However, no polyglucose granules such as those found in D. gigas [START_REF] Hensgens | Electron-dense granules in Desulfovibrio gigas do not consist of inorganic triphosphate but of a glucose pentakis(diphosphate)[END_REF] could be detected by electronic microscopy in the ES strains (data not shown).

According to the genome sequencing results, only a limited number of mutations accounted for the O 2 respiratory growth of the ES5 and ESE strains. To determine whether the absence of the GEI and/or inactivation of the hdrA gene was sufficient to confer this new capability to the ES5 strain, the growth behaviours of both the DvH GEI - [START_REF] Johnston | A genomic Island of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough promotes survival under stress conditions while decreasing the efficiency of anaerobic growth[END_REF] and its derived ΔhdrA deletion mutant strains were studied in the presence of oxygen. As shown in Fig. 1C, the DvH GEI -strain was unable to grow in pyruvate/sulfate-free medium under continuous 0.65% O 2 -sparging, while the GEI -ΔhdrA strain grew, although more slowly than the ES5 strain. These observations suggest that in the GEI -genetic context, the inactivation of HdrA is critical to permit the cells to respire oxygen for growth. However, this mutation is not sufficient alone to stabilize this metabolic capability over successive subculturings, suggesting that, in addition, fine metabolic tuning is required.

Since HdrA was critical for oxygen respiration in the ES5 strain, the expression level of hdrA in the ESE cells was also determined. Surprisingly, the hdrA gene was upregulated [14.4 AE 3.4 times (mean AE SD, n = 9)] when the ESE cells where cultured with O 2 compared with WTOR. The importance of HdrA in the ESE cells was examined by deleting the gene that encodes it. A significant loss of growth capability was observed when the ESEΔhdrA strain was grown in pyruvate/sulfate-free medium under continuous 0.65% O 2sparging compared with the ESE strain (Fig. 1D). However, the growth behaviours of the ESE and ESEΔhdrA strains were highly similar when cultured in lactate/sulfate-limiting medium under continuous O 2 -sparging (data not shown). Thus, HdrA seems to be critical for O 2 respiration when pyruvate is used as an electron donor but not with lactate. These results agree with data indicating that HdrA is more important for pyruvate-based than lactate-based energy metabolism [START_REF] Meyer | The energy-conserving electron transfer system used by Desulfovibrio alaskensis strain G20 during pyruvate fermentation involves reduction of endogenously formed fumarate and cytoplasmic and membrane-bound complexes, Hdr-Flox and Rnf[END_REF][START_REF] Price | The genetic basis of energy conservation in the sulfatereducing bacterium Desulfovibrio alaskensis G20[END_REF][START_REF] Ramos | The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris Hildenborough[END_REF].

Mutations upstream of the tetR gene were detected in the genome of both the ES5 and ESE strains. Compared with WTOR, the expression of tetR, as determined by qPCR, was slightly higher in both the ES5 and ESE strains cultured with O 2 [3.7 AE 0.5 and 3.0 AE 0.6 times (mean AE SD, n = 9) respectively]. Thus, the mutations identified upstream of the tetR gene induced a change in its expression that would contribute to the metabolic variation in the ES strains. Transcriptomic analysis is required to identify additional transcriptional variations in the ES strains.

Discussion

Sulfate-reducing bacteria, especially Desulfovibrio species, are among the most aerotolerant anaerobic microorganisms and have been subjected to numerous studies to identify the molecular and cellular mechanisms involved in their aerotolerance capability. While the response of Desulfovibrio species to a transient oxidative stress is well documented, only a few studies examining their growth under continuous exposure to oxidative conditions have been reported [START_REF] Johnson | Oxygen-dependent growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough[END_REF][START_REF] Ramel | Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases[END_REF]. In this study, via O 2 -driven experimental evolution, Desulfovibrio vulgaris Hildenborough strains, evolved to grow under continuous 0.65% oxygensparging corresponding to 7.7 μM of dissolved oxygen at 33 C, were obtained. This concentration is 32-fold greater than that tolerated by the ancestral DvH strain [START_REF] Ramel | Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases[END_REF]. In addition to their increased aerotolerance, we have unambiguously demonstrated that the O 2 -evolved strains are capable of a true oxygen respiratory process that leads to oxidative phosphorylation and permits growth. The oxidation of molecular hydrogen or organic compounds coupled to oxygen reduction has been demonstrated in several Desulfovibrio species [START_REF] Dannenberg | Oxidation of H 2 , organic compounds and inorganic sulfur compounds coupled to reduction of O 2 or nitrate by sulfate-reducing bacteria[END_REF]. Proton gradient formation and energy production with hydrogen and oxygen as an electron donor and acceptor, respectively, have already been shown in Desulfovibrio desulfuricans [START_REF] Fitz | A study on electron transport-driven proton translocation in Desulfovibrio desulfuricans[END_REF][START_REF] Cypionka | Oxygen respiration by Desulfovibrio species[END_REF]. However, oxygen reduction was never associated with growth. Weak growth (no more than one doubling) of Desulfovibrio desulfuricans and Desulfobacterium autotrophicum with O 2 as an electron acceptor has been reported, but it was accompanied by a rapid loss of cell motility and viability [START_REF] Marschall | Influence of oxygen on sulfate reduction and growth of sulfatereducing bacteria[END_REF]. Recently, it has been observed that the magnetotactic bacterium Desulfovibrio magneticus forms a band of cells at the oxic-anoxic interface in semi-solid oxygengradient medium [START_REF] Lefevre | Growth of magnetotactic sulfate-reducing bacteria in oxygen concentration gradient medium[END_REF]. Thus, the authors proposed that this bacterium used oxygen as an electron acceptor, but they did not provide an accurate characterization. An aerobic lifestyle has been proposed for the cable bacteria, members of the anaerobic Desulfobulbacceae family. These multicellular filamentous bacteria have been shown to mediate long-distance (centimetre scale) electron transport from sulfide to oxygen in both marine and freshwater sediments [START_REF] Schauer | Succession of cable bacteria and electric currents in marine sediment[END_REF][START_REF] Risgaard-Petersen | Cable bacteria in freshwater sediments[END_REF]. Electrons are generated from sulfide oxidation in cells at one end, pass through internal insulated wires to cells at the other end where they are used to reduce oxygen [START_REF] Pfeffer | Filamentous bacteria transport electrons over centimetre distances[END_REF]. However, this atypical aerobic lifestyle raises interesting questions about energy conservation and cell differentiation, since sulfide oxidation and oxygen reduction do not occur in the same cell. Consequently, efficient growth linked to oxygen respiration in a pure culture of any SRB has never been clearly demonstrated before. Therefore, the O 2 -evolved strains obtained and characterized in this study represent the first example of a Desulfovibrio species exhibiting an energy metabolism based on oxygen respiration that confers the capability of efficient and perennial true O 2 respiratory growth. This new capability was obtained after a limited number of generations (114 generations) and a few genomic mutations. Two distinct genotypes were obtained: a major genotype characterized by the absence of the GEI as well as a frameshift mutation in the hdrA gene and an insertion in the regulatory sequence of the tetR encoding gene, and a minor genotype characterized by the presence of the GEI and a deletion in the regulatory expression sequence of the tetR encoding gene. These genotypes resulted from both the selection of a pre-existing polymorphism in the ancestral DvH and the occurrence of new mutations that rapidly arose after a low number of generations. Polymorphic loci and a rapid selective sweep of pre-existing mutations within the first 100 generations followed by the slow fixation of new mutations has been observed in the experimental evolution of D. vulgaris Hildenborough under salt stress for up to 5000 generations [START_REF] Zhou | Rapid selective sweep of preexisting polymorphisms and slow fixation of new mutations in experimental evolution of Desulfovibrio vulgaris[END_REF][START_REF] Zhou | Key metabolites and mechanistic changes for salt tolerance in an experimentally evolved sulfatereducing bacterium[END_REF]. The authors identified 11 mutations within the first 1200 generations [START_REF] Zhou | Rapid selective sweep of preexisting polymorphisms and slow fixation of new mutations in experimental evolution of Desulfovibrio vulgaris[END_REF] and an additional 25 after 5000 generations, as well as large deletions in both the chromosome and the plasmid [START_REF] Zhou | Key metabolites and mechanistic changes for salt tolerance in an experimentally evolved sulfatereducing bacterium[END_REF]. Even if the experimental evolution under O 2 selective pressure reported here had been conducted for a much lower generation number, mutation selection from pre-existing polymorphisms (i.e., the DVU2402 mutation) and genome reduction (i.e., loss of the GEI) can highlight a common phenomenon in adaptive evolution.

DvH took two distinct routes of evolution to acquire the capability of O 2 respiration. The first, as represented by the ES5 strain, primarily involved the inactivation of the HdrA protein and the loss of the GEI, while the second, represented by the ESE strain, was associated with the overexpression of the hdrA gene. The hdrA gene (DVU2402) is part of the hdr-flx operon, which encodes the flavin oxidoreductase FlxABCD and the heterodisulfide reductase HdrABC [START_REF] Heidelberg | The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough[END_REF][START_REF] Ramos | The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris Hildenborough[END_REF]. In D. alaskensis G20, the Flx-Hdr complex is involved in pyruvate-based energy metabolism [START_REF] Meyer | The energy-conserving electron transfer system used by Desulfovibrio alaskensis strain G20 during pyruvate fermentation involves reduction of endogenously formed fumarate and cytoplasmic and membrane-bound complexes, Hdr-Flox and Rnf[END_REF]. This complex bifurcates electrons from NADH to DsrC, part of the dissimilatory sulfite reductase DsrABC, and to ferredoxin, allowing an increased yield of reduced ferredoxin, which can be used by an ion-pumping ferredoxin:NADH oxidoreductase (Rnf ) to pump ions into the periplasm and participate in ion gradient generation [START_REF] Meyer | The energy-conserving electron transfer system used by Desulfovibrio alaskensis strain G20 during pyruvate fermentation involves reduction of endogenously formed fumarate and cytoplasmic and membrane-bound complexes, Hdr-Flox and Rnf[END_REF][START_REF] Price | The genetic basis of energy conservation in the sulfatereducing bacterium Desulfovibrio alaskensis G20[END_REF]. The Flx-Hdr complex can also operate in reverse in DvH, reducing NAD + to regenerate oxidized ferredoxin [START_REF] Ramos | The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris Hildenborough[END_REF]. Therefore, the central role of HdrA in the pyruvate-based O 2 respiratory growth for the ES strains is highlighted in Fig. 4.

In the ES5 strain (Fig. 4A), because of the absence of a functional Flx-Hdr complex, the NADH generated would primarily be re-oxidized by the membrane-bound NADH oxidase, which, in turn, would reduce the menaquinone pool [START_REF] Ramel | Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation[END_REF]. Menaquinol would be then oxidized by the bd quinol oxidase, which reduces O 2 in water and thus contributes to the proton motive force. The absence of the GEI, which includes the gene encoding the NADH-dependent ROO1 that permits cytoplasmic O 2 reduction without energy conservation, would enhance the efficiency of this O 2 respiratory process by increasing the reduced NADH pool available for membrane-bound oxygen reduction. In the ESE strain (Fig. 4B), the hdr-flx operon is overexpressed, and thus, the Flx-Hdr complex would operate in reverse, regenerating oxidized ferredoxin and reducing NAD + that, in turn, will be used by the bd quinol oxidase for O 2 respiration as described above. However, phenotypic analyses have shown that the absence of the GEI and hdrA mutation are not the unique factors involved, and even if they appeared early during the laboratory evolution experiment (19 generations), a fine-tuning of metabolism must have occurred during the continuation of the experiment (till 114 generations), probably in relationship with TetR, to make the O 2 respiratory process as efficient and perennial for growth as the sulfate respiration.

These ES strains are the first sulfate-reducing bacteria that exhibit both true oxygen and sulfate respiratory processes. The O 2 -driven experimental evolution of DvH led to a lifestyle change of this anaerobic microorganism, allowing it to switch from sulfate to oxygen respiratory growth. This capability, acquired after only a limited number of mutations, should be related to the presence of all enzymatic systems required for both oxygen and sulfate respiration in DvH, and we propose that a rearrangement of metabolite fluxes, especially that of NAD + /NADH, ultimately enables the optimization of O 2 respiration to sustain growth. The data presented in this study provide clues to understand the metabolic versatility of microorganisms at the boundary between aerobic and anaerobic lifestyles.

Experimental procedures

Bacterial growth conditions

The culture media were based on medium C as previously described [START_REF] Postgate | The genomes of Desulfovibrio gigas and D. vulgaris[END_REF] but varied depending on the carbon source and sulfate concentration. Lactate/sulfate medium contained 37 mM lactate and 32 mM sulfate while lactate/sulfate-limiting medium contained 37 mM lactate and 9 mM sulfate. Sulfate-free medium was prepared the same way as above except that sodium sulfate was omitted and MgCl 2 and FeCl 2 were used to replace the corresponding metal sulfate salts. In this medium, no sulfate could be detected by ion chromatography. Pyruvate (40 mM) was used instead of lactate when indicated. All the strains were cultured at 33 C in Hungate tubes containing 10 ml of medium. All the strains were stored in liquid nitrogen from cultures grown in lactate/sulfate medium. For each condition, two successive cultures were performed before analysing the phenotype. Cultures were either grown under anaerobic conditions or under continuous O 2 -sparging conditions with a gas flow rate of 40 ml min -1 at various oxygen concentrations provided by a gas mixer (PEGAS 4000 MF, Columbus Instruments, USA) as previously described [START_REF] Ramel | Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases[END_REF]. Growth in pyruvate/sulfate-free medium was performed either under anaerobic conditions or under continuous N 2 sparging (40 ml min -1 ) to lower the hydrogen partial pressure in the culture [START_REF] Martins | Desulfovibrio vulgaris growth coupled to Formate-driven H 2 production[END_REF]. The absence of contamination in the cultures were regularly checked by wet mount microscopy as well as by streaking culture aliquots onto a Luria-Bertani agar plate followed by an incubation at 37 C. The dissolved oxygen concentration was monitored using a Mettler-Toledo M700 recorder equipped with an O 2 module ppb 4700 and a calibrated Inpro 6900 O 2 probe. The growth was monitored by measuring the optical density at 600 nm. The growth rates were estimated by fitting the scatter plots of the logarithmic optical density at 600 nm from at least four independent experiments to a linear regression. The number of cells per volume unit of culture medium was determined using a Thoma cell counting chamber as previously described [START_REF] Ramel | Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases[END_REF].

Experimental evolution

For the O 2 -driven experimental evolution of DvH, the WTOR strain was successively cultured at 33 C in lactate/sulfate medium under continuous O 2 -sparging conditions at increasing oxygen concentrations (Supporting Information Fig. S1). Briefly, an anaerobic culture in lactate/sulfate medium of a clonal isolate of the ancestral DvH strain (WTOR) was used to inoculate 10 ml of fresh lactate/sulfate medium in Hungate tubes under continuous 0.02% O 2 -sparging conditions. Cultures were incubated at 33 C until the OD 600nm reached its maximum (early stationary phase) and used to inoculate new fresh lactate/sulfate medium sparged with a higher oxygen concentration. At least two successive cultivations under the same oxygenation condition were performed. The oxygen concentrations used were 0.02%, 0.04%, 0.08%, 0.12%, 0.16%, 0.20%, 0.25%, 0.35%, 0.45%, 0.55% and 0.65%. All the cultures were inoculated at 20% (v/v). For the anaerobic-driven experimental evolution, the same successive culturing procedure was applied, except that all the cultures were maintained under anaerobic conditions. Subculturing was stopped when the same number of generations as the O 2 -driven experimental evolution was reached. Samples of the intermediates and final evolved cultures were frozen and stored in liquid nitrogen for further analysis. Clones were isolated from the evolved populations by plating on solid E medium [START_REF] Postgate | The genomes of Desulfovibrio gigas and D. vulgaris[END_REF] at 33 C in an anaerobic chamber (COY) under a 10% H 2 /90% N 2 mixed gas atmosphere. Whole-genome sequencing Genomic DNA was prepared from 10 ml cultures in lactate/sulfate medium. The cells were harvested by centrifugation at 5700g for 15 min at 4 C. Total DNA was extracted from the cell pellets using a Wizard Genomic DNA purification kit (Promega) according to the manufacturer's instructions. Illumina sequencing of the total DNA, as well as data analysis to detect potential SNPs and InDels, were conducted on either a GATC Biotech AG (Germany) or a Beckman Coulter Genomics (USA) platform. The genome of DvH (sequence NC_002937.3) was used as a reference.

ATP measurement and oxygen consumption activity in whole cells

The intracellular ATP concentration was measured using an Intracellular ATP kit (Biothema) according to the manufacturer's instructions. Cultures were grown to mid-log phase in pyruvate/sulfate medium under anaerobic conditions or in pyruvate/sulfate-free medium under continuous 0.65% O 2 -sparging. A 20 μL aliquot of mid-log phase culture previously diluted 100-fold was deposited into a black 96 well plate (CORNING™ 3915), and the reagents were added as indicated by the manufacturer. Luminescence was recorded using a Luminoskan Ascent luminometer (Thermo Scientific), and the data were analysed using the 2.6 version of Ascent software (Thermo Scientific).

Oxygen consumption activity in whole cells

Cultured cells in lactate/sulfate-limiting medium (80 ml) were centrifuged at 5700g at 4 C for 15 min. The pellet was resuspended in 40 ml of 150 mM NaCl, 100 mM Tris-HCl buffer (pH 7.5) (buffer A) previously flushed with N 2 to eliminate oxygen and centrifuged again. This washing step was repeated five times to eliminate any trace sulfur that would react with oxygen. Oxygen consumption in the whole cells was measured at 30 C in a 2.3 mL stirred microrespiration chamber filled with air-saturated buffer A using an oxygen microsensor (OX-100) (Unisense). Cells (typically 1.6 mg of protein) were first added to the chamber, and lactate was added at a final concentration of 90 mM. The oxygen concentration was recorded using BasicSensor trace software (Unisense). The protein concentrations of the cell suspensions were determined using a Pierce BCA protein assay kit (ThermoScientific) after the addition of 1 M NaOH to disrupt the cells.

End-product analysis

Samples of cultures (600 μl) were collected immediately after inoculation and at the end of growth and centrifuged for 10 min at 13 400 g (4 C). The supernatants were frozen in liquid nitrogen and stored at -20 C until used. Lactate, pyruvate and acetate were quantified using highperformance liquid chromatography (HPLC) as previously described [START_REF] Ramel | Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases[END_REF]. The sulfate concentration was measured on an ion chromatograph (761 Compact IC, Metrohm) equipped with an anion exchange column (Metrosep A Supp1 250 × 4.6 mm). Na 2 CO 3 (3 mM) was used as the mobile phase at a flow rate of 1 ml min -1 during the first 8 min, followed by a flow rate of 2.5 ml min -1 . The culture supernatants were diluted 10-fold before injection (20 μl). Detection of sulfite and thiosulfate was performed on the same system. Hydrogen sulfide was quantified spectrophotometrically according to the Cline method [START_REF] Cline | Spectrophotometric determination of hydrogen sulfide in natural waters[END_REF].

TEM analyses

Cultures were grown to mid-log phase in lactate/sulfatelimiting medium under anaerobic conditions or continuous 0.65% O 2 -sparging. Cells were collected by centrifugation (3000 g, 5 min, ambient temperature) and resuspended in 100 mM Tris buffer (pH 8). Five microliters of the bacterial suspension were placed directly on glow discharged carbon coated grids (EMS) for 3 min. The grids were washed with two drops of 2% aqueous uranyl acetate and stained with a third drop for 1 min. The grids were dried on filter paper, and the samples were analysed using a Tecnai 200KV electron microscope (FEI), and a numeric camera (Eagle, FEI) was used to acquire digital images. The images recorded were then treated using ImageJ software.

Quantitative real-time PCR RNA was prepared from 5 ml cultures at mid-log phase after the addition of 5 ml of RNAlater (Thermo Fisher Scientific). The cells were harvested by centrifugation (5700 g for 15 min at 4 C). The cell pellets were frozen in liquid nitrogen and stored at -80 C until use. Total RNA was prepared using a Maxwell ® 16 LEV simplyRNA Blood Kit (Promega) according to the manufacturer's instructions with an extra TURBO DNase (ThermoFisher) digestion step to eliminate any contaminating DNA. The RNA quality was checked using an Experion™ StdSens Kit (Bio-Rad), and the absence of DNA contamination was confirmed by PCR. Nucleic acids were quantified spectrophotometrically at 260 nm (NanoDrop 2000C ThermoScience). For cDNA synthesis, 1 μg of total RNA and 0.5 μg of random primers (Promega) were used with GoScript™ Reverse transcriptase (Promega) according to the manufacturer's instructions. Quantitative-real time PCR (qPCR) were performed as previously described [START_REF] Vita | The primary pathway for lactate oxidation in Desulfovibrio vulgaris[END_REF].

The number of copies of roo1 (DVU2014) and roo2 (DVU3185) were determined by qPCR on genomic DNA prepared from 10 ml cultures as described above. Pure DvH genomic DNA was used as the standard, and a technical duplicate was performed for each point. The amplification efficiencies for each primer pair ranged between 75% and 100%. All the primer pairs used for qPCR are reported in Supporting Information Table S3.

Construction of the ΔhdrA deletion mutant strains

The deletion vector pnot19Δhdra, based on the pNOTCmΔ [START_REF] Vita | The primary pathway for lactate oxidation in Desulfovibrio vulgaris[END_REF] vector backbone, was constructed by the direct transformation of PCR products into E. coli [START_REF] You | Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis[END_REF] using specific primers pairs described in Supporting Information Table S3. It was then transferred to either the DvH GEI -or ESE strain by electro-transformation as previously described [START_REF] Vita | The primary pathway for lactate oxidation in Desulfovibrio vulgaris[END_REF]. After electro-transformation, the cell/DNA mixture was transferred to 10 ml of lactate/sulfate-limiting medium in Hungate tubes and cultured at 33 C for 5 h before the addition of thiamphenicol (30 μg ml -1 ). Deletion mutant strains were isolated as described above and the hdrA deletion genotype was PCR verified.

Fig. 1 .

 1 Fig. 1. Growth curves of the O 2 -evolved culture (circle), obtained after successive subcultures with stepwise increases of oxygen concentration for 114 generations, and anaerobic-evolved culture (square), obtained after successive subcultures under anaerobic conditions for 114 generations, in lactate/sulfate medium under continuous 0.65% O 2 -sparging (A), of ES5 (circle), ESE (square), 114G2 (triangle) and WTOR (diamond) strains in pyruvate/sulfatefree medium under continuous 0.65% O 2 -sparging (B). Growth curves of ES5 (circle), DvH GEI -(cross), GEI -ΔhdrA (diamonds) strains (C), as well as ESE (square) and ESEΔhdrA (triangle) strains (D) in pyruvate/sulfate-free medium under continuous 0.65% O 2 -sparging. The growth curves of the isolated strains were obtained from at least four independent experiments. Error bars are the SDs in the measurements.

Fig. 2 .

 2 Fig. 2. Quantification of the copies of the roo1 (DVU2014) gene relative to the roo2 (DVU3185) gene by qPCR. The numbers of copies of roo1 are normalized to 1E + 06 copies of roo2. The percentages indicated correspond to the oxygen concentration applied during the course of the O 2 -driven evolution. Error bars are the SDs in the measurements (n = 3). WTOR corresponds to the ancestral DvH strain, and 114G corresponds to the anaerobic-evolved culture after 114 generations. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology

Fig. 3 .

 3 Fig. 3. Intracellular ATP concentration of the cells grown in pyruvate/ sulfate medium under anaerobic conditions (white bars) or in pyruvate/sulfate-free medium under continuous 0.65% O 2 -sparging (grey bars). Concentrations were determined in three independent cell preparations. Error bars are the SDs in the measurements. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology

Fig. 4 .

 4 Fig. 4. The central role of HdrA in the pyruvate-based O 2 respiratory growth for ES5 (A) and ESE (B) strains. BD: BD quinol oxidase, Rnf: ferredoxin:NADH oxidoreductase complex, MK/MKH2: menaquinol pool, HdrABC: heterodisulfide reductase complex, FlxABCD: flavin oxidoreductase, Fdred/ox: reduced/oxidized ferredoxin, dsrC: dissimilatory sulfite reductase C. Thick arrows correspond to major fluxes and dashed arrows to minor fluxes.

©

  2018 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology O 2 -driven experimental evolution of Desulfovibrio 9

Table 1 .

 1 Mutations identified in O 2 -evolved ESC, D, E, ES3, 4 and 5 strains and anaerobic-evolved strains 114G1, 2 and 3 strains compared with the ancestral DvH (WTOR) strain used for evolution.

		Mutation	Mutation		Genomic	Nucleotide	Amino-acid
	Strain	type	position	Affected gene(s)	localization	change	change
	ESC/D/E	Deletion	Intergenic	DVU1690, tetR, transcriptional regulator	1 768 101	TAACA à T	Non-coding (-75)
		Deletion	Gene	DVU3134, glpK, glycerol kinase	3 282 104*	TC à T	-82 amino acids
		SNP a	Gene a	DVU0171, patB, hemolysin related protein a	213 843 a	A à G a	Val à Ala a
	ES3/4/5	Insertion	Gene	DVU2402, hdrA, heterodisulfide reductase A	2 507 946*	T à TAG	-514 amino acids
		Deletion	Gene	DVU3134, glpK, glycerol kinase	3 281 432*	AG à A	-282 amino acids
		Insertion	Intergenic	DVU1690, tetR, transcriptional regulator	1 768 033	Duplication of region	Non-coding (-143)
						1 768 033 to
						1 768 116
		SNP b	Gene b	DVU0126, ABC transporter b	159 851 b	T à G b	Leu à Arg b
	114G1/2/3	Insertion	Intergenic	DVU0435, hypothetical protein	489 733*	G à GA	Non-coding (+9)
		SNP	Gene	DVU0681, sensor histidine kinase	755 786	C à T	Ala à Thr
		SNP	Intergenic	DVU1941, HAD superfamily hydrolase	2 014 879*	G à A	Non-coding (-13)
		SNP	Gene	DVU1941, HAD superfamily hydrolase	2 014 855*	G à A	Asp à Asp
		Insertion	Gene	DVU3134, glpK, glycerol kinase	3 281 945*	T à TGGTA	-181 amino acids
		SNP c	Gene c	DVU1469, ribosomal protein S1 c	1 551 147 c	C à T c	Gly à Arg c
		Deletion c	Gene c	DVU3134, glpK, glycerol kinase c	3 281 991 c *	C G Aà C c	-92 amino acids c
	Grey highlights mutations common within ESC/D/E, ES3/4/5 or 114G1/2 strains.

(nb)

corresponds to the localization of the mutation when intergenic compared with the start codon (-nb) or compared with the stop codon (+nb). (-XXX) amino acids corresponds to the number of suppressed amino acids due to the deletion. Abbreviation: SNP: single nucleotide polymorphism.

Table 2 .

 2 Specific growth rate (μ) and maximal OD 600nm for the ancestral DvH WTOR strain, anaerobic-evolved 114G2 strain and O 2 -evolved ES5 and ESE strains cultured in different conditions.

	Pyruvate/sulfate-free	μ (h -1 ) 600nm Max. OD	ng ng	0.077 AE 0.001 AE 0.11 1.06	ng ng	0.074 AE 0.02 AE 0.13 1.08	ng ng	ng ng
	Pyruvate/sulfate	μ (h -1 ) Max. OD 600nm	0.088 AE 0.01 1.38 AE 0.01	np np	0.131 AE 0.01 1.29 AE 0.06	np np	0.141 AE 0.01 1.49 AE 0.01	0.139 AE 0.02 0.57 AE 0.01
	Lactate/sulfate-limiting	μ (h -1 ) Max. OD 600nm	0.108 AE 0.004 0.78 AE 0.01	0.132 AE 0.005 1.35 AE 0.05	0.115 AE 0.014 0.71 AE 0.02	0.142 AE 0.01 1.28 AE 0.02	0.098 AE 0.002 0.72 AE 0.02	0.152 AE 0.003 0.79 AE 0.02
	Medium Lactate/sulfate	Strain Condition a μ (h -1 ) Max. OD 600nm	ES5 -O 2 0.098 AE 0.06 1.21 AE 0.04	+O 2 0.099 AE 0.016 1.22 AE 0.01	ESE -O 2 0.146 AE 0.028 1.15 AE 0.01	+O 2 0.147 AE 0.014 1.19 AE 0.04	WTOR -O 2 0.084 AE 0.008 1.18 AE 0.02	114G2 -O 2 0.093 AE 0.001 1.22 AE 0.02	np: not performed; mean AE SD (at least n = 4).	ng: no growth during the time period of 40 h.	a Aeration condition of cultures. +O 2 corresponds to a continuous 0.65% O

-sparging, -O 2 corresponds to anaerobiosis. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology

© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology

Acknowledgements

The authors thank G. Voordouw for providing the DvH GEI - strain. This study benefited from the facilities and expertise of the Platform for Microscopy of IMM (A. Kosta and H. Le Guenno). The authors do not have any conflict of interest to declare.

Supporting Information

Additional Supporting Information may be found in the online version of this article at the publisher's web-site: Fig. S1. Experimental evolution process. Apparatus for growth in the presence of defined O 2 concentrations (A). Experimental design to establish the desired continuous O 2 -sparging (B). Illustration of the experimental workflow (C). A clonal isolate of the ancestral DvH (WTOR) strain was grown in lactate/sulfate medium (medium C). This culture was then used to initiate the evolution by inoculating it in medium C under 0.02% O 2 -sparging. The O 2 concentration was successively increased during the course of evolution, reaching an O 2 concentration of 0.65% after 114 generations (114 g). For the anaerobicdriven experimental evolution, the same successive culturing procedure was applied under anaerobic conditions. At the end of both evolutionary periods, clones were isolated from the evolved population. Genome of six O 2evolved (ESC, D, E and ES3, 4 and 5) and 3 anaerobicevolved clones (114G1, 2 and 3) and the WTOR strain were sequenced. Among them, characterization was conducted on the ESE, ES5 and 114G2 strains. Fig. S2. Confirmation of mutations in the intergenic region of Dvu1690 (tetR gene): PCR products of the intergenic region upstream DvU1690 (tetR gene) using the couple of primers DVU1690Left and DVU1690Right (see Table S3) using as template the evolved population at various steps of the O 2 -driven experimental evolution as well as the ES5 and ancestral DvH (WTOR) strains. Lane M: size markers (A). Sanger sequencing of the region corresponding to the deletion in the intergenic region of tetR (see Table ) Culture were performed in lactate (37 mM)/sulfate (9 mM) medium under anaerobic conditions or under continuous 0.65% O 2 sparging (+O 2 ) with (+MoO 4 ) or without (-MoO 4 ) the addition of 250 μM MoO 4 (A). Culture were performed in pyruvate (40 mM)/sulfate (9 mM) medium under anaerobic conditions or in pyruvate (40 mM)/sulfate-free medium under continuous 0.65% O 2 sparging (+O 2 ) with (+MoO 4 ) or without (-MoO 4 ) the addition of 250 μM MoO 4 (B). At the end of the growth, cells were collected, and the protein concentration was determined. Table S1. NGS data of the sequenced genome strains. Table S2. Mutations identified in DvH WTOR strain compared to the NCBI D. vulgaris Hildenborough reference sequence NC_002937.3 Table S3. Oligonucleotide primers used.

© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology