Olivier N Lemaire

Flora A Honoré

Sébastien Tempel

Emma M Fortier

Silke Leimkühler

Vincent Méjean

Chantal Iobbi-Nivol

Shewanella decolorationis LDS1 chromate resistance

The Shewanella genus is well known for its genetic diversity, its outstanding respiratory capacity and its high potential for bioremediation. Here, a novel strain isolated from sediments of the Indian Ocean was characterized. 16S rRNA analysis indicated that it belongs to the S. decolorationis species. It was named Shewanella decolorationis LDS1. This strain presented an unusual ability to grow efficiently from 24 °C to 40 °C, without apparent modifications of its metabolism as shown by testing respiratory activities or carbon assimilation and in a wide range of salt concentrations. Moreover, S. decolorationis LDS1 tolerates high chromate concentrations. Indeed, it was able to grow in the presence of 4 mM chromate at standard temperature and 3 mM at 40 °C. Interestingly, whatever the temperature, when the culture reached the stationary phase, the strain reduced the chromate present in the growth medium. In addition, S. decolorationis LSD1 degrades different toxic dyes including anthraquinone-, triarylmethane-and azo-dye. Thus, compared to S. oneidensis, this strain presented better capacity to cope with various abiotic stresses in particular at high temperatures. The analysis of genome sequence preliminary data indicated that, in contrast to S. oneidensis and S. decolorationis ST2, S. decolorationis LDS1 possesses the phosphorothioate modification machinery that has been described to participate against various abiotic stresses by protecting DNA. We demonstrate that its heterologous production in S. oneidensis allows it to resist higher concentrations of chromate. decolorationis LDS1 is highly attractive since its metabolism allows it to develop efficiently from 24 to 40 °C conserving its ability to respire alternative substrates and to reduce toxic compounds such as chromate or toxic dyes. Our results clearly indicate that this novel strain has the potential to be a tool for bioremediation and unveil one of the mechanisms involved in the chromate resistance.

Importance

Shewanella species have long been described as interesting microorganisms in regards to their ability to reduce many organic and inorganic compounds including metals. However, members of the Shewanella genus are often depicted as cold water microorganisms although usually their optimal growth temperature ranges around 25-28 °C under laboratory growth conditions. S.

Introduction

Shewanella species are part of the gammaproteobacteria class of the Proteobacteria phylum.

They have been found all around the world in briny or fresh water and sediments from polar circles to equatorial biotopes [START_REF] Hau | Ecology and biotechnology of the genus Shewanella[END_REF][START_REF] Nealson | Ecophysiology of the Genus Shewanella[END_REF]. Their trade mark is their tremendous variety of respiratory capacities allowing their growth with a large panel of exogenous electron acceptors including solid and soluble metals, explaining why they belong to the restricted family of dissimilatory metal-reducing bacteria [START_REF] Fredrickson | Towards environmental systems biology of Shewanella[END_REF]. Partly due to their reducing abilities, Shewanella species have the capacity to cope with elevated concentrations of toxic products as for example hydrocarbons, arsenate and hexahydro-1,3,5-trinitro-1,3,5-triazine [START_REF] Gao | Microbial diversity and abundance in the Xinjiang Luliang long-term water-flooding petroleum reservoir[END_REF][START_REF] Saltikov | The ars detoxification system is advantageous but not required for As(V) respiration by the genetically tractable Shewanella species strain ANA-3[END_REF][START_REF] Sibanda | Synthetic extreme environments: overlooked sources of potential biotechnologically relevant microorganisms[END_REF][START_REF] Suganthi | Enhanced biodegradation of hydrocarbons in petroleum tank bottom oil sludge and characterization of biocatalysts and biosurfactants[END_REF][START_REF] Sun | Characterization of the microbial community composition and the distribution of Femetabolizing bacteria in a creek contaminated by acid mine drainage[END_REF][START_REF] Zhao | Shewanella halifaxensis sp. nov., a novel obligately respiratory and denitrifying psychrophile[END_REF]. This ability has made them a useful tool for bioremediation and clean-up (1-3, 10, 11). Moreover, it appears that the members of this genus adapt easily to various life conditions. Although they are usually found in aquatic habitats, some can develop in other biotopes like butter, crude oil or even human corpses [START_REF] Hau | Ecology and biotechnology of the genus Shewanella[END_REF][START_REF] Nealson | Ecophysiology of the Genus Shewanella[END_REF][START_REF] Kakizaki | Detection of bacterioplankton in immersed cadavers using selective agar plates[END_REF]. Within the genus, S. oneidensis MR-1 has become the strain of reference in many laboratories and it is clearly the best described. As most of the other species, its optimal growth temperature is around 25-28 °C [START_REF] Venkateswaran | Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp[END_REF] and it requires the HtpG general chaperone to survive at 35 °C [START_REF] Honoré | Hsp90 Is Essential under Heat Stress in the Bacterium Shewanella oneidensis[END_REF]. It can grow in either brackish or fresh water and it possesses an efficient chemosensory system that allows it to adapt rapidly under nutrient deprivation or stressful conditions [START_REF] Bouillet | The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis[END_REF][START_REF] Bouillet | Protection of the general stress response σS factor by the CrsR regulator allows a rapid and efficient adaptation of Shewanella oneidensis[END_REF][START_REF] Liu | Transcriptome Analysis of Shewanella oneidensis MR-1 in Response to Elevated Salt Conditions[END_REF]. It was shown that S. oneidensis MR-1 survives to elevated concentrations of chromate by developing several resistance strategies like oxidative stress protection, detoxification, SOS-controlled DNA repair mechanisms and different pathways for Cr(VI) reduction [START_REF] Brown | Molecular dynamics of the Shewanella oneidensis response to chromate stress[END_REF][START_REF] Viamajala | Chromate/nitrite interactions in Shewanella oneidensis MR-1: evidence for multiple hexavalent chromium [Cr(VI)] reduction mechanisms dependent on physiological growth conditions[END_REF]. Indeed, in addition to enzymatic reduction of chromate (hexavalent chromium Cr(VI)) into a less mobile form (Cr(III)) that presents a decreased toxicity [START_REF] Barnhart | Chromium chemistry and implications for environmental fate and toxicity[END_REF][START_REF] Meng | Iron(III) minerals and anthraquinone-2,6-disulfonate (AQDS) synergistically enhance bioreduction of hexavalent chromium by Shewanella oneidensis MR-1[END_REF][START_REF] Myers | Chromium (VI) reductase activity is associated with the cytoplasmic membrane of anaerobically grown Shewanella putrefaciens MR-1[END_REF][START_REF] Viamajala | Chromate reduction in Shewanella oneidensis MR-1 is an inducible process associated with anaerobic growth[END_REF], S. oneidensis, as other members of the genus, possesses a chromate efflux pump able to extrude chromate from the cell increasing its resistance against the metal [START_REF] Baaziz | ChrASO, the chromate efflux pump of Shewanella oneidensis, improves chromate survival and reduction[END_REF].

Although, S. oneidensis is the model bacterium of the genus, many other species are studied because, beside their common characteristics, they present peculiar aspects conferring useful properties especially for biotechnology [START_REF] Hau | Ecology and biotechnology of the genus Shewanella[END_REF][START_REF] Nealson | Ecophysiology of the Genus Shewanella[END_REF][START_REF] Fredrickson | Towards environmental systems biology of Shewanella[END_REF][START_REF] Zhong | Pan-genome analyses of 24 Shewanella strains re-emphasize the diversification of their functions yet evolutionary dynamics of metal-reducing pathway[END_REF]. Thus, these bacteria often have been isolated from polluted areas, waste water or sewage plants. Recently, the genome of Shewanella algidipiscicola H1, isolated from the sediment of the industrial Algerian Skikda harbor, was sequenced [START_REF] Baaziz | Draft Genome Sequence of Shewanella algidipiscicola H1, a Highly Chromate-Resistant Strain Isolated from Mediterranean Marine Sediments[END_REF]. This strain is proposed to be the most resistant Shewanella in chromiumcontaining medium. A second strain highly resistant to toxic metals, Shewanella ANA-3 comes from water contaminated with arsenate that the bacterium uses as a respiratory substrate [START_REF] Saltikov | The ars detoxification system is advantageous but not required for As(V) respiration by the genetically tractable Shewanella species strain ANA-3[END_REF].

Shewanella halifaxensis, isolated from an immersed munition-dumping zone, can degrade explosive compounds to generate respiratory substrates (9). As a last example, Shewanella decolorationis S12 capable of efficient dye-decolorization activity was isolated from sludges of a textile-printing waste-water treatment plant in China [START_REF] Xu | Shewanella decolorationis sp. nov., a dye-decolorizing bacterium isolated from activated sludge of a waste-water treatment plant[END_REF]. Thus, in the Shewanella genus, the various species present distinctive properties that make them genuine tools to cope with a large range of human environmental misconducts. This large panel of enzymatic activities is correlated to the genetic variability found in the Shewanella genus and the latter was difficult to define correctly because of the diversity of the species it contains [START_REF] Hau | Ecology and biotechnology of the genus Shewanella[END_REF][START_REF] Nealson | Ecophysiology of the Genus Shewanella[END_REF][START_REF] Fredrickson | Towards environmental systems biology of Shewanella[END_REF][START_REF] Zhong | Pan-genome analyses of 24 Shewanella strains re-emphasize the diversification of their functions yet evolutionary dynamics of metal-reducing pathway[END_REF]. This point is illustrated by the increasing number of Shewanella species found in databases and corresponding to almost 70 species and 30 sequenced genomes.

The high diversity of Shewanella genomes is really astonishing. Recently, pan-genome analyses of 24 Shewanella strains established that the core genome corresponds to only 12% of the total genes [START_REF] Zhong | Pan-genome analyses of 24 Shewanella strains re-emphasize the diversification of their functions yet evolutionary dynamics of metal-reducing pathway[END_REF]. This small number of shared genes is consistent with a genus that is genetically versatile and has had frequent lateral gene transfer events.

The purpose of the current study is to characterize a novel strain, S. decolorationis LDS1 to gain greater insight into the Shewanella genus behaviors in bioremediation and depollution.

Since S. decolorationis LDS1 reduced chromate into a less soluble and thus less toxic compound, decolorized various dyes, both in a large range of temperatures, and possessed a DNA modification system that is suspected to protect bacteria against environmental stresses, this novel strain presents an interesting biotechnological potential.

Results and Discussion

Strain isolation and characterization.

S. decolorationis strain LDS1 (for La Digue Seychelle and also named S. decolorationis sesselensis, see accession number) was isolated from a sample of sediments collected in a pond at the Anse Coco beach (La Digue Island, Republic of Seychelles, Indian Ocean) on the basis of its capability to grow on rich medium containing trimethylamine oxide (TMAO). Indeed, the latter is a compound widespread in marine environments, and with a few exceptions, it is used as a major respiratory substrate by Shewanella species [START_REF] Santos | Molecular analysis of the trimethylamine N-oxide (TMAO) reductase respiratory system from a Shewanella species[END_REF][START_REF] Lemaire | Efficient respiration on TMAO requires TorD and TorE auxiliary proteins in Shewanella oneidensis[END_REF][START_REF] Lemaire | Small membranous proteins of the TorE/NapE family, crutches for cognate respiratory systems in Proteobacteria[END_REF][START_REF] Yancey | Marine fish may be biochemically constrained from inhabiting the deepest ocean depths[END_REF]. Based on a comparison of the conserved 16S rRNA genes of over thirty Shewanella species, a phylogenetic tree was constructed (Figure 1). The Shewanella genus can be divided into two types, the mesophilic and the piezotolerant species [START_REF] Banerjee | Niche specific amino acid features within the core genes of the genus Shewanella[END_REF][START_REF] Kato | Correlation between phylogenetic structure and function: examples from deep-sea Shewanella[END_REF]. S. decolorationis LDS1 was among mesophilic subgroup in the clade containing the two other defined strains of S. decolorationis species with which it shared 100 % 16S rRNA gene identity [START_REF] Xu | Shewanella decolorationis sp. nov., a dye-decolorizing bacterium isolated from activated sludge of a waste-water treatment plant[END_REF][START_REF] Chen | Biodegradation of crystal violet by a Shewanella sp. NTOU1[END_REF]. These species known for their decolorization activities are described to grow at higher temperatures than S. oneidensis and most other species, since they survive at 40 °C and present an optimal growth from 20 to 30 °C.

Since Shewanella species are originally marine bacteria (2), S. decolorationis LDS1 was cultivated at various salt concentrations. It was able to grow from very low to high concentrations of salt, even higher than that of the ocean (about 0.5 M, Figure 2A) [START_REF] Liu | Transcriptome Analysis of Shewanella oneidensis MR-1 in Response to Elevated Salt Conditions[END_REF]. S. decolorationis LDS1 was then phenotypically characterized in order to test its ability to grow at high temperature and to decolorize dyes, and thus to explore if it exhibits different features compared to the already known strains of the clade. The strain was cultivated at various temperatures from 24 to 42 °C and the exponential growth rates are presented in Figure 2B. As the other S. decolorationis strains [START_REF] Xu | Shewanella decolorationis sp. nov., a dye-decolorizing bacterium isolated from activated sludge of a waste-water treatment plant[END_REF], S. decolorationis LDS1 could grow at more elevated temperatures than most of the other Shewanella species and, although less efficient, growth still occurred at 42 °C. Interestingly, in contrast to the other Shewanella species described, S. decolorationis LDS1 showed a uniquely wide optimal growth temperature since it presented a plateau ranging from 24 to 40 °C. S. decolorationis LDS1 was then challenged for its ability to decolorize Remazol Brilliant Blue, Crystal violet and Congo red, which are members of the three families of dyes: anthraquinone, triarylmethane and azo dye, respectively. As expected for the strains of this species, the dyes were at least partially converted by S. decolorationis LDS1 at 28 °C (Figure 2C). We used this property to check the efficiency of the strain at 40 °C, and as shown in Figure 2C, S. decolorationis LDS1 was still able to decolorize the three families of dyes. At both temperatures, the color of the media without bacteria remained identical for more than ten days (Figure 2C). This strain seems to be able to decolorize dyes including anthraquinone-, triarylmethane-and azo-dye from 28 to 40 °C. This characterization suggests that S. decolorationis LDS1 presents an adapted metabolism that allows it to cope efficiently from medium to elevated temperatures.

To better determine the physiological capacities of the strain at high temperature, we decided to compare its TMAO reductase activity at 28 and 40 °C. The TMAO reductase activity was first followed through the alkalization of the growth medium indicating that the increase of the temperature has no effect since the alkalization of the medium was similar (Table 1). For a more accurate activity, we measured the TMAO reductase activity of crude extracts of cells grown at 28 or 40 °C. The TMAO reductase specific activity of this strain at 40 °C was half that at 28 °C (2.8±0.17 µmol/min/mg versus 5.95±0.40 µmol/min/mg, respectively). Measures of nitrate reduction and other phenotypic characteristics obtained with API20NE plates (Biomerieux, France) are recorded in Table 1 and showed no significant difference at 28 and 40 °C, except for the hydrolysis of capric acid that could be due to the instability of the product rather than the activity of the enzyme. Altogether these results suggest that the strain has fully adapted from medium to elevated temperatures by increasing the range of its optimal growth temperature.

The tested respiratory activities (oxygen, TMAO, nitrate) confirmed that they were efficient from 28 to 40 °C. In contrast to S. decolorationis S12T and the other strains presented in Table 2, S. decolorationis LDS1 could not grow at very low temperature. The fact that this strain seemed to have lost the latter ability was not surprising considering that the biotope from where it was isolated probably does not encounter temperature below 25 °C.

Other differences could be pointed out. Indeed, counter to the S. decolorationis S12T, S. decolorationis LDS1 as many other Shewanella species did not use glucose for fermentation although it performed its assimilation (Table 2). Moreover, the range of optimal pH for growth ranged from 6.0 to 11.0, which was slightly wider compared to that of other Shewanella strains [START_REF] Xu | Shewanella decolorationis sp. nov., a dye-decolorizing bacterium isolated from activated sludge of a waste-water treatment plant[END_REF]. This probably results from the sampling of S. decolorationis LDS1 from brackish water that can be more acidic than sea water. Altogether, these results indicate that the S. decolorationis LDS1 shares with other S. decolorationis strains the ability to grow at higher temperatures than most of the other Shewanella species. However, only S. decolorationis LDS1 presents the same growth rate from 24 to 40 °C. Moreover, it appears that its metabolism is not drastically affected by high temperature since most of the tested activities were similar or still measurable whatever the growth temperature.

Chromate resistance and reduction.

Recent studies have focused on the ability of Shewanella species to reduce chromate [START_REF] Viamajala | Chromate/nitrite interactions in Shewanella oneidensis MR-1: evidence for multiple hexavalent chromium [Cr(VI)] reduction mechanisms dependent on physiological growth conditions[END_REF][START_REF] Meng | Iron(III) minerals and anthraquinone-2,6-disulfonate (AQDS) synergistically enhance bioreduction of hexavalent chromium by Shewanella oneidensis MR-1[END_REF][START_REF] Myers | Chromium (VI) reductase activity is associated with the cytoplasmic membrane of anaerobically grown Shewanella putrefaciens MR-1[END_REF][START_REF] Viamajala | Chromate reduction in Shewanella oneidensis MR-1 is an inducible process associated with anaerobic growth[END_REF][START_REF] Baaziz | ChrASO, the chromate efflux pump of Shewanella oneidensis, improves chromate survival and reduction[END_REF]. For instance, S. oneidensis MR-1 can resist 1 mM chromate; whereas, S. algidipiscicola H1 grows in 3 mM of chromate [START_REF] Brown | Molecular dynamics of the Shewanella oneidensis response to chromate stress[END_REF][START_REF] Baaziz | Draft Genome Sequence of Shewanella algidipiscicola H1, a Highly Chromate-Resistant Strain Isolated from Mediterranean Marine Sediments[END_REF]. In order to compare the resistance against chromate of S. decolorationis LDS1 with that of the model bacterium S. oneidensis, a preliminary test was done on solid medium containing a filter soaked with a 1 M-chromate solution, leading to the formation of a gradient of chromate by diffusion in the solid medium. As shown in Figure 3A, S. decolorationis LDS1 was more resistant than S. oneidensis at 28 °C since it was able to grow closer to the filter. Moreover, in liquid medium, S. oneidensis barely resisted 1 mM chromate [START_REF] Brown | Molecular dynamics of the Shewanella oneidensis response to chromate stress[END_REF] while this concentration did not affect S. decolorationis LDS1 (Figure 3B). This indicates that S. decolorationis LDS1 can resist higher chromate concentrations. To confirm this point, its growth was followed under aerated conditions at 28 °C in the presence of increasing concentrations of chromate. As shown in Figure 3B, no drastic growth difference was noticeable in the range of chromium concentration from 0 to 2 mM, although a short lag phase was observed at 2 mM. In the presence of 3 and 4 mM chromate, the cell growth presented a pronounced lag phase of about 20 hours, but finally the curves reached the same plateau value in comparison to the cultures grown at lower concentrations or without the addition of chromate. This long lag phase could be necessary for the bacteria to adapt to the high chromate concentrations by producing either Cr(VI) reducing systems or Cr efflux pumps. In addition, the chromate resistance was followed at 40 °C. The results indicate that the growth is not modified until 1 mM of chromate in the medium. At higher concentrations, the lag phases were increased (15 and 30 hours for 2 and 3 mM, respectively) while at 4 mM no growth was observed after 40 hours. Altogether these data establish that S. decolorationis LDS1 resists chromate even at elevated temperatures.

Finally, we wondered whether S. decolorationis LDS1 was able to reduce chromate during its growth. This is of great interest since chromate reduction leads to a less soluble and less toxic form for the environment [START_REF] Barnhart | Chromium chemistry and implications for environmental fate and toxicity[END_REF][START_REF] Viamajala | Chromate reduction in Shewanella oneidensis MR-1 is an inducible process associated with anaerobic growth[END_REF]. Thus, S. decolorationis LDS1 was grown at 28 °C, in the presence of 1 mM of chromate and reduction of Cr(VI) to Cr(III) in the growth medium was followed. Strikingly, when the medium was inoculated with cells of S. decolorationis LDS1, the Cr reduction appeared when the culture entered into the stationary phase (Figure 4). From that time, the reduction of chromate was almost complete within 15 hours. A similar experiment was performed at 40 °C. As for the lower temperature, the reduction of chromate started when the cells reached the stationary phase (Figure 4). From these results, it is clear that S. decolorationis LDS1 can reduce chromate at high temperature with the same efficiency as at 28 °C. The mechanism involved is clearly acting from the beginning of the stationary phase as suggested by the trigger of the reduction when the bacteria reach it, as already described under anaerobic growth of S. oneidensis MR-1 [START_REF] Viamajala | Chromate reduction in Shewanella oneidensis MR-1 is an inducible process associated with anaerobic growth[END_REF].

Data from genome sequence.

Since S. decolorationis LDS1 was able to cope better with various stresses than does S. oneidensis and has a better heat resistance than S. decolorationis S12T, we wondered whether this strain has specific machineries leading to an increase of its resistance to environmental cues. Indeed, Shewanella species are described to present a high level of genetic variability that confers to each of them peculiar properties [START_REF] Fredrickson | Towards environmental systems biology of Shewanella[END_REF][START_REF] Zhong | Pan-genome analyses of 24 Shewanella strains re-emphasize the diversification of their functions yet evolutionary dynamics of metal-reducing pathway[END_REF]. The genome of S. decolorationis LDS1 was rsequenced at the Molecular Research LP (MR DNA) Laboratory (USA). A preliminary analysis indicated that the draft genome sequence contained 4223 potential protein-coding genes. A genome comparison was undertaken between the two S. decolorationis strains and S. oneidensis MR-1 (Table 3). Among genes shared by the two other species, more than 40 code for c-type cytochromes, which are a trade mark of bacteria of Shewanella genus. However, the results indicated that 12% of S. decolorationis LDS1 genome was not shared by the two other strains. Among the 259 potential genes present only in S. decolorationis LDS1 genome, 153 have been annotated but 99 of them have an unknown function (Table 4). From the other 54 genes, several of them drew our attention (Table 4). For instance, we noticed two operons that could play a central role in the adaptation of the strain to environmental stresses. First, the dndBCDE operon that encodes the phosphorothioate (PT) modification machinery that replaces a nonbridging oxygen by a sulfur in the DNA sugar phosphate backbone (35-37) (Table 4).

The sequences recognized by the modification machinery are not fully established and studies have shown that only 12 % of the potential sites recognized by the PT system are effectively modified and from one cell to another, the modifications are not conserved [START_REF] Cao | Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences[END_REF][START_REF] Tong | Occurrence, evolution, and functions of DNA phosphorothioate epigenetics in bacteria[END_REF]. Moreover, it seems that when the potential target site is present in the binding motif of an important regulatory protein, no modification occurs. This was demonstrated for TorR and RpoE binding sites [START_REF] Cao | Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences[END_REF]. In Shewanella species the Tor system, which is regulated by TorR, is a major respiratory system [START_REF] Bordi | Genes regulated by TorR, the trimethylamine oxide response regulator of Shewanella oneidensis[END_REF] and RpoE controls the expression of stress response genes [START_REF] Barchinger | Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation[END_REF].

Generally, the modification machinery includes an additional protein, DndA, a cysteine desulfurase [START_REF] Xu | DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus[END_REF]. The dndA gene is absent in S. decolorationis LDS1 and, as described in literature, it is probably replaced in the machinery by IscS (SDECO_v1_40139) [START_REF] Tong | Occurrence, evolution, and functions of DNA phosphorothioate epigenetics in bacteria[END_REF]. A bioinformatics analysis using DndC sequence as a probe indicated that only three unrelated species of Shewanella, S. baltica, S. pealeana ATCC 700345 and S. violacea DSS12 possess this machinery with 92.8%, 67.8% and 66.5% of identity, respectively. This probably indicates that S. decolorationis LDS1 has acquired the genes of the Dnd machinery from another bacterial genus. pthFGH, the other interesting operon [START_REF] Xu | A novel host-specific restriction system associated with DNA backbone S-modification in Salmonella[END_REF], is found in the same locus as the dndBCDE operon.

It is related to the dnd gene products and it encodes DNA restriction enzymes, and they form together the modification-restriction (M-R) system. In many bacteria producing the Dnd machinery, the restriction system is absent [START_REF] Cao | Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences[END_REF]. The inability of S. decolorationis LDS1 to grow at low temperature is noticeable. Indeed, it was established in E. coli B7A and in Salmonella enterica serovar Cerro 87 that the restriction system is temperature dependent and is activated at 15 °C [START_REF] Yang | DNA Backbone Sulfur-Modification Expands Microbial Growth Range under Multiple Stresses by its anti-oxidation function[END_REF]. The temperature dependence of the restriction system in S. decolorationis LDS1 is yet to be determined as well as the role of that restriction system once activated.

Protection by the DndBCDE machinery.

The function of this M-R system is not well understood and an additional role in the epigenetic control of gene expression is suspected [START_REF] Tong | Occurrence, evolution, and functions of DNA phosphorothioate epigenetics in bacteria[END_REF]. Indeed, the products of the dnd operon were shown to sustain bacterial growth under stresses like H2O2, heat or salinity by protecting DNA from oxidative damage and restriction enzymes [START_REF] Yang | DNA Backbone Sulfur-Modification Expands Microbial Growth Range under Multiple Stresses by its anti-oxidation function[END_REF]. In agreement with experiment done in Escherichia coli and S. piezotolerans, our hypothesis is that the production of DndB-E proteins improves the growth of S. decolorationis LDS1 under oxidative conditions. The presence of chromate leads to oxidative damages in bacteria. Thus, to investigate whether the dnd operon plays a role in the robustness of S. decolorationis LDS1 towards chromate, dndBCDE genes carried by the pBAD33 vector (pdndBCDE) were introduced into S. oneidensis MR-1. S. oneidensis MR-1 harboring either pdndBCDE or pBAD33 and S. decolorationis LDS1 were grown aerobically in the presence of arabinose to induce the dndBCDE expression from the plasmid, and serial dilutions were spotted on plates containing chromate (0.4 mM) (Figure 5).

The growth of S. oneidensis MR-1 harboring pBAD33 was drastically affected, whereas when carrying pdndBCDE, the strain was able to grow. Growth of S. decolorationis LDS1 was similar in the presence or absence of chromate. This result indicates that the presence and expression of the dndBCDE operon allow S. oneidensis to resist high chromate concentrations.

However, since S. decolorationis LDS1 resist better and also higher concentrations, we suggest that the DndBCDE machinery is only one element of the bacterial equipment allowing the high level of resistance of S. decolorationis LDS1. To ascertain the effect due to the expression of dndBCDE operon in S. oneidensis MR1, the reverse experiment was done and increased chromate concentration were spotted on a lawn of bacteria. As shown on figure 5B, the presence of dndBCDE genes led to a better resistance of the strain after 24-and 48h incubation.

Moreover, we tested whether the DndBCDE machinery led S. oneidensis MR-1 to develop at temperatures higher than 28 °C. At both 38 and 40 °C, the growth of S. oneidensis MR-1 was not improved by the presence of DndBCDE suggesting that this machinery is not involved in the temperature resistance.

Conclusion.

In conclusion, the novel S. decolorationis LDS1 strain is an intriguing organism since it is adapted to higher temperature than most of the other members of this genus. Moreover, it keeps its enzymatic capacity in a large range of elevated temperatures and under these conditions, its metabolism seems not perturbed. Indeed, its ability to degrade the three families of dyes tested in this study is conserved even at high temperature. We can hypothesize that S. decolorationis LDS1 probably degrades other pollutants. Its capacity to tolerate high concentrations of chromate in the culture medium indicates that S. decolorationis LDS1 has developed strategies to cope with chromate toxicity. Indeed, from the results obtained here, it appears that during exponential growth, the strain resists the toxic effect of chromate until stationary phase is reached, thus at high cell concentration, when the reduction of chromate occurs. The features of S. decolorationis LDS1 that it grows efficiently from 24 to 40 °C and it reduces both toxic dyes and chromate likely makes it an efficient tool for bioremediation of many pollutants.

Material and methods

Isolation and genome sequencing

The S. decolorationis LDS1 strain was isolated from a sample of the uppermost 3 centimeters of muddy sediment of a brackish pound from the Anse Coco Beach in La Digue island (Seychelles Republic, 4°21'56.309"S 55°51'4.367"E). The sample was conserved in sterile plastic tube kept a room temperature during expedition. The sediments were suspended 24 hours in 15 mL of a 1:4 dilution of LB (1.25 g yeast extract, 1.25 g NaCl, 2.5 g tryptone per liter) pH 7.2 in the presence of 40 mM of TMAO and 0.5 M NaCl in closed plastic tubes and at room temperature (around 20 °C). To avoid alkalinization of the medium due to trimethylamine production, 50 mM of MOPS pH 7.2 were added. The upper part of the culture, mud-less due to the sedimentation, was submitted to repeated enrichments in similar medium and growth conditions. Among the different colony morphotypes of isolates on agar plates, a red-brownish colony was selected for characterization and conserved at -80 °C in LB medium with 20 % (vol/vol) glycerol.

After DNA purification, the genome sequencing of S. decolorationis LDS1 was carried out at the "Molecular Research LP (MR DNA) Laboratory" (USA). The library was prepared with Nextera DNA Sample Preparation Kit (Illumina) following the manufacturer's user guide and sequenced with the HiSeq 2500 system (Illumina) as previously described [START_REF] Baaziz | Draft Genome Sequence of Shewanella algidipiscicola H1, a Highly Chromate-Resistant Strain Isolated from Mediterranean Marine Sediments[END_REF].

Bacterial strains and growth conditions.

The S. oneidensis strain used in this study correspond to the rifampicin resistant MR-1-R [START_REF] Bordi | Anticipating an alkaline stress through the Tor phosphorelay system in Escherichia coli[END_REF] and was routinely grown at 28 °C in LB medium under agitation. When necessary, chloramphenicol (25 μg/mL) was added. The S. decolorationis LDS1 strain was routinely grown at 28 °C in the same medium.

When required, media were solidified by adding 17 g.L -1 agar. When necessary, TMAO (40 mM) and FeCl3 (1 mM) were added in the medium as alternative electron acceptors. When mentioned, in the presence of TMAO, MOPS pH 7.2 (25 mM) was added to buffer the growth medium and avoid alkalization by trimethylamine production. Bacterial growth on plates: Strains containing either the pBAD33 vector or pdndBCDE plasmid were grown overnight at 28°C in LB medium amended with chloramphenicol (25 μg/mL). S. decolorationis LDS1 was grown under the same conditions except that no chloramphenicol was added. They were then diluted to OD600=0.1 in fresh LB medium and were incubated at 28°C. At OD600=0.7, arabinose (0.2%) was added for 2 hours to induce dndBCDE expression.

Cells were then diluted to OD600=1 and 2 µL drops of 10-fold serial dilutions were spotted on LB-agar plates that contained arabinose (0.2%) in the presence or absence of chromate (0.4 mM), but no chloramphenicol. The plates were incubated at 28 °C.

Physiological characterization

The growth temperature and salinity ranges were assessed in a Tecan Spark 10M microplate reader in 24 well transparent plates under agitation with a humidity cassette to avoid dehydration. For salinity range determination, growths were carried out at 28 °C. During exponential phase, the exponential growth rates were calculated. Growth at 4 °C was tested on agar plates, no growth was detected after two months. Phenotypic characteristics were determined with API 20NE test strips (Biomérieux), incubated at 28 or 40 °C. TMAO reduction was assessed by the measure of pH during growth in unbuffered LB medium in the presence of TMAO and spectroscopically at 30 °C on crude extracts of the strain grown at 28 or 40 °C as described in 29. Iron reduction was evaluated by black precipitates formation during growth in the presence of FeCl3.

Dye decolorization

Decolorization assays were performed in static aerated plastic tubes incubated at 28 or 40 °C.

Sterile LB medium was amended with 0.001 % crystal violet (Sigma Aldrich), 0.005 % Congo Red (Sigma Aldrich) or 0.005 % Remazol Brilliant Blue (Sigma Aldrich). These media were either inoculated with S. decolorationis LDS1 preculture in order to reach an OD600 of 0.2 or not inoculated (negative control) and incubation was carried out during ten days. For the inoculated media, cells were removed by centrifugation and remaining medium was transferred in a 24 well transparent plate for imaging.

Chromate resistance and reduction

Chromate solutions were made by dilution of potassium chromate K2CrO4 (Sigma Aldrich) in deionized water.

For chromate resistance assay on agar plate, 150 µL of bacterial culture at OD600 of 0.001 were spread out on LB agar plate. A sterile cotton patch containing 50 µL of potassium chromate 1 M solution was placed at the center of the plate. Diffusion of the solution in the agar forms a chromate gradient. Lengths of the bacteriostatic halo around the patch for each strain were compared after incubation at 28 °C for 12 hours.

For chromate resistance assay on agar plates, recombinant S. oneidensis MR1 were first grown at 28 °C until late exponential phase and then, induced for 2 h in the presence of arabinose (0.2 %). 400 µL of bacterial culture diluted at OD600 of 0.01 were spread out. After a 2 h-incubation, three sterile cotton patches containing 25 µL of potassium chromate (5, 10, 20 mM) were placed on the plates. Diffusion of the solution in the agar forms a chromate gradient. Lengths of the bacteriostatic halo around the patch for the two strains were compared after 24-and 48-hour incubation at 28 °C.

Chromate resistance in liquid culture was assayed in LB medium in the presence of 0, 0.5, 1, 2, 3 or 4 mM of potassium chromate. OD600 was measured in a Tecan Spark 10M microplate reader in 24 well transparent plates under agitation at 28 or 40 °C and with a humidity cassette to avoid dehydration.

Chromate concentration was measured by the 1,5-diphenyl carbazide (DPC, Sigma Aldrich) method slightly modified [START_REF] Baaziz | ChrASO, the chromate efflux pump of Shewanella oneidensis, improves chromate survival and reduction[END_REF][START_REF] Li | Selective, peroxidase substrate based "signal-on" colorimetric assay for the detection of chromium (VI)[END_REF]. Briefly, hexavalent chromate was detected in culture supernatant by the colorimetric reaction of a solution of 0.025 % (vol/vol) DPC, 0.5 % (vol/vol) H3PO4 in deionized water. After 5-to 10-minute reaction, optical density at 540 nm was measured. Chromate concentration was calculated by comparison to a standard range.

Phylogenetic tree construction

A classical phylogeny pipeline was used from the phylogeny tool MEGA7 [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets[END_REF]. The sequences were aligned with MUSCLE [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]. The evolutionary history was inferred from the Neighbor-Joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]. The tree with the highest log likelihood (-4930.1497) is shown (200 replicates). The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method [START_REF] Tamura | Prospects for inferring very large phylogenies by using the neighbor-joining method[END_REF] and are in the units of the number of base substitutions per site. The analysis involved 33 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1128 positions in the final dataset.

Homologous genes

BLAST version 2.6.0 (51) was used to determine if genes from genome A are homologous to genes from genome B. Two genes were considered homologous if the e-value hit was lower or equal to 1e -5 . The BLAST results and a R script shows the genes present in one, two or the three Shewanella genomes.

pdndBCDE construction

To construct the pdndBCDE plasmid, the dnd operon from S. decolorationis LDS1 chromosome with its Shine Dalgarno sequence was PCR-amplified with forward and reverse primers containing the XmaI and SphI restriction sites, respectively. After digestion with XmaI and SphI, the DNA fragment was inserted into the pBAD33 vector digested with the same restriction enzymes. The resulting plasmid was checked by DNA sequencing using several internal primers and then introduced into MR1-R by conjugation. of 33 species of Shewanella. The tree with the highest log likelihood (-4930.1497) is shown.

The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 33 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1128 positions in the final dataset.

Figure 2 .

 2 Figure 2. S. decolorationis LDS1 behaviours

Figure 3 .

 3 Figure 3. Chromate resistance of S. S. decolorationis LDS1 and S. oneidensis MR-1

Table 2 . Phenotypic characteristics of various Shewanella species

 2 . Species (27, 52): 1, S. decolorationis LDS1; 2, S. decolorationis S12T; 3, S. baltica DSM 9439; 4, S. glacialipiscicola NBRC 102030T; 5, S. hafniensis; 6, S. oneidensis ATCC 700550T; 7, S. putrefaciens ATCC 8071. ND, not determined.

Table 3 .

 3 Genome overview of S. decolorationis LDS1, S.

decolorationis S12T and S. oneidensis MR-1.

		S. decolorationis	S. decolorationis	S. oneidensis
		LDS1	S12T	MR-1
	GC content	47.12	47.09	45.96
	size	4719362	4851566	4969811
	DNA scaffolds	16	77	0
	Total genes	4430	4451	5031
	Coding sequences	4223	4309	4444
	Common genes		3535
	Strain-specific genes	259	345	375

Table 4 . Proteins specifically found in S. decolorationis LDS1 from genome sequencing and annotation.

 4

Acknowledgements

We would like to thank Olivier Genest, Michel Fons and the whole BIP03 team for interesting discussions. We are grateful to Scot E. Dowd (Molecular Research LP [MR DNA] Laboratory) for sequencing and assembly of our genome. The LABGeM (CEA/IG/Genoscope and CNRS UMR8030) and the France Génomique National infrastructure (funded as part of the Investissement d'avenir program managed by the Agence Nationale pour la Recherche, contract ANR-10-INBS-09) are acknowledged for allowing access to the MicroScope platform. This work was supported by the Centre National de la Recherche Scientifique (CNRS) and Aix-Marseille Université (AMU). O.N. L. was supported by a MESR fellowship, AMU and CNRS.

We thank the Aix-Marseille Université for supporting S.L. with a visiting professor position.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Data availability

The results obtained from the whole-genome shotgun project have been deposited at the National Center for Biotechnology Information (NCBI) under accession number SHEWANELLA_DECOLORATIONIS_SESSELENSISCP037898.

A. Chromate resistance was tested on solid LB medium. 150 µl of cells (OD= 0.001) were spread on the plate and 50 µl of chromate (1 M) was loaded on the filter. Plates were incubated 12 hours at 28 °C. The radius (R) of each halo is indicated in cm.

B. and C. Chromate resistance of S. decolorationis LDS1 was tested on liquid LB medium at 28 and 40 °C with increasing concentration of chromate: 0 mM (light blue), 0.5 mM (orange), 1 mM (grey), 2 mM (yellow), 3 mM (dark blue) and 4 mM (green). Each point corresponds to the average of at least three independent replicates. Averages and standard deviation from at least three independent experiments are shown.


Biljana Mitrova

Armel F T Waffo

Paul Kaufmann

Chantal Iobbi-Nivol

Silke Leimkühler

Ulla Wollenberger

Electrochemical biosensor for TMAO detection with a chimeric enzyme

Keywords: Trimethylamine N-oxide (TMAO), TMAO reductase, chimeric enzyme, molybdoenzyme, electrochemical biosensor

For the first time, an enzyme-based electrochemical biosensor system for determination of trimethylamine N-oxide (TMAO) is described. It employs an active chimeric variant of TorA in combination with an enzymatically deoxygenating system and a low potential mediator for effective regeneration of the enzyme and cathodic current generation.TMAO reductase (TorA) is a molybdoenzyme found in marine and most enterobacteria that catalyzes specifically the reduction of TMAO to trimethylamine (TMA). The chimeric TorA named TorA-FDH corresponds to the apoform of TorA from Escherichia coli reconstituted with the molybdenum cofactor from formate dehydrogenase (FDH). Each enzyme, TorA and TorA-FDH, was immobilized on the surface of a carbon electrode and covered with a dialysis membrane. The biosensor operates at an applied potential of -0.8 V (vs. Ag/AgCl (1M KCl)) under ambient air conditions thanks to an additional enzymatic O 2 -scavenger system. A comparison between the two enzymes revealed a much higher sensitivity for the biosensor with immobilized TorA-FDH. The biosensor exhibits a sensitivity of 14.16 nA/µM TMAO in a useful measuring range of 2 -110 µM with a detection limit of LOD = 2.96 nM (n = 3), almost similar for TMAO in buffer and in spiked serum samples. With a response time of 16 ± 2 s the biosensor is stable over prolonged daily measurements (n=20). This electrochemical biosensor provides suitable applications in detecting TMAO levels in human serum.

Introduction

Trimethylamine N-oxide (TMAO) is an organic end-product originating from the degradation of dietary trimethylamine containing compounds like choline, carnitine and lecithin (Velasquez et al., 2016). After ingestion of food rich in these compounds the gut microbiota degrades them to trimethylamine (TMA), which is further transferred to the liver where the flavin dependent monooxygenase (FMO) family of enzymes converts it to TMAO. This TMAO is mostly excreted through urine (Fennema et al., 2016). In recent years this molecule has emerged as a potential biomarker for several disease states. Clinical reports have pointed to the involvement of TMAO in the development of atherosclerosis (Bennett et al., 2013) and its correlation to the development and progression of cardiovascular diseases (Wang et al., 2011). A link between high levels of TMAO in the plasma and the increased risk of developing colorectal cancer among postmenopausal women was also found (Bae et al., 2014). Given the main clearance of TMAO from the body is through urine (Smith et al., 1994), increased negative effects have also been reported in patients with chronic kidney disease (Bell et al., 1991;Stubbs et al., 2016).

Even though the emerging importance of the TMAO as a biomarker has been recognized

there is yet no specific method for the detection of this molecule that could be used quickly outside the laboratory. Most of the reported methods in literature are analytical approaches that first need the reduction of TMAO to TMA (daCosta et al., 1990) or some other preparation methods that involve organic toxic solvents and labeling. In some cases, an additional derivatization of the TMA is also needed in order to get an estimate of the TMAO levels. Methods like ion or liquid chromatography and nuclear magnetic resonance (NMR) were mostly used in the developed procedures for determining the TMAO levels (Bell et al., 1991;Mamer et al., 2010;Wang et al., 2014). Apart from the reported unspecific microbial biosensor (Gamati et al., 1991) a specific electrochemical biosensor approach, to our knowledge, is not yet developed.

Most of the biosensors available on the market are based on oxidases and operate at positive potentials. In contrast, the use of reductase reactions usually requires potentials where dissolved oxygen interferes and therefore, it has to be removed beforehand. The combination of glucose oxidase and catalase is well established for deoxygenation of solutions for various analytical approaches (Benesch and Benesch, 1953;Cheng et al., 2003;Englander et al., 1987). This approach was elaborated in great detail by Plumeré et al. for oxygen tolerant biosensing of nitrate (Plumeré et al., 2012). Previously, an amperometric DMSO biosensor on the basis of DMSO reductase, a highly homologous enzyme to TMAO reductase was reported where application of glucose oxidase and catalase enabled measurements of DMSO in ambient air conditions (Abo et al., 2003).

Trimethylamine N-oxide reductase, TorA, from Escherichia coli is a mononuclear molybdenum containing enzyme which specifically catalyzes the reduction of TMAO to TMA in the reaction (Buc et al., 1999):

TMAO + 2H + + 2e - TMA + H 2 O
It can also catalyze other N-oxide compounds, like 4-methyl morpholine N-oxide as an example of another prominent substrates (Iobbi-Nivol et al., 1996). Among the reported substrates, TMAO and morpholine N-oxide have the highest affinity for TMAO reductase and are catalyzed also with the highest efficiency. Recently, Kaufmann et al. reported the reconstitution of an active TMAO reductase from the apoform of the enzyme (apo-TorA) with the cofactor from Rhodobacter capsulatus formate dehydrogenase (FDH). Direct and mediated electrochemistry established that high electrocatalytic activities were achieved when the enzyme was either expressed and purified under anaerobic conditions (referred to as TorA-WT) or reconstituted with the FDH cofactor ligating a terminal sulfido ligand at the molybdenum atom (referred to as TorA-FDH) (Kaufmann et al., 2018).

Here, we report the first electrochemical TMAO biosensor. The sensor is based on TMAO reduction by immobilized TorA-FDH in combination with an enzymatically deoxygenation system and a low potential mediator for effective regeneration of the engineered enzyme. All of the measurements were performed with the chimeric enzyme TorA-FDH that is exhibiting high catalytic activity. A comparison between TorA-FDH or TorA-WT biosensor demonstrates that TorA-WT is less efficient than the chimeric enzyme TorA-FDH. Given the higher signals obtained with mediated electrochemistry compared to direct electrochemistry, we chose the first one for the development of the TMAO biosensor. The glucose oxidase/catalase system ensured an O 2 insensitive detection of TMAO by the biosensor at ambient air condition.

Experimental

Apparatus and procedures

The electrochemical experiments were carried out in a lab-made three-electrode cell with a total volume of 1 mL, employing a platinum wire as counter electrode, an Ag/AgCl (1 M KCl) reference electrode (Microelectrodes Inc., Bedford, USA) against which all potentials are reported. Cyclic voltammetry (CV) was performed with a modified graphite rod working electrode (Alfa Aesar, Karlsruhe, Germany). A glassy carbon electrode (BAS) was employed as the working electrode in all amperometric measurements for the development of the biosensor. All electrochemical experiments were performed at room temperature using PalmSens potentiostat and analyzed with PSLite 4.8 software (Palmsens, The Netherlands)

and OriginPro 2017. The CV experiments were performed in a glovebox (COY, USA) at room temperature and under anaerobic conditions (minimum of 2% H 2 and 98% N 2 ).

In CV, the potential on the working electrode was cycled between -0.2 V and -0.8 V for the direct bioelectrocatalysis and between -0.3 V and -0.9 V for the mediated bioelectrocatalysis, all at 5 mV/s scan rate and stirred solution. In amperometry, the potential applied on the working electrode was held at constant value of -0.8 V.

The biosensor characterization was performed under ambient air conditions using 8 U GOx and 200 U Cat of final activity together with 50 mM glucose to insure O 2 removal from the measuring cell. A lid on top of the cell was placed to limit the O 2 dissolving into the measuring buffer during the measurement.

Electrode modification

CV measurements

Prior to modification the graphite rod electrode with diameter of 3.

Protein purification and reconstitution

The purification of E. coli TorA, apo-TorA and the reconstitution of apo-TorA with the cofactor of R. capsulatus formate dehydrogenase were performed as described in Kaufmann et al. (Kaufmann et al., 2018).

Results and discussion

Direct versus mediated bioelectrocatalysis

In a recent study, we reported on the direct electrocatalytic reaction of TorA and the in vitro reconstituted chimera of this enzyme in order to indicate the role of the first coordination sphere of the cofactor on its potential and its activity (Kaufmann et al., 2018). TorA-FDH was among the most active enzyme variants. (Fourmond et al., 2013). This potential should be close to the redox potential of the center where catalysis takes place or/and to the redox potential of the center which is a limiting factor during the electron transfer event (Zu et al., 2003). Given that TMAO reductase carries only the redox center bis-MGD, we can ascribe the measured catalytic potential to represent a close value of the potential of the redox active center. Then, a similar experiment was conducted except that a mediator was added (Fig 1B). Methyl viologen was selected mostly because of its negative redox potential (E° = -0.446 V vs. NHE (i.e., = -0.645 V vs. Ag/AgCl (1 M KCl)) (Michaelis and Hill, 1933). As

shown in Fig 1b, the addition of TMAO in the presence of methyl viologen leads to a strong current increase. Indeed, the bioelectrocatalytic current is more than 80 times higher than in the unmediated bioelectrocatalysis. The efficiency of the electron transfer is obviously much higher in the presence of mediator. In mediated electrochemistry, the molecules which are not in direct contact with the electrode also contribute to the total biocatalytic current output. This makes the use of a mediator a much more desirable approach for the construction of biosensors where small concentrations of substrates should be detected.

Biosensor

The previous voltammetric experiments were conducted in a glovebox in order to prevent the interferences coming from oxygen. The biosensor is intended to be applicable under standard, environmental conditions (in ambient air) in the lab. Therefore, the buffer solution was enzymatically deoxygenated during the measurements and glassy carbon was used instead of graphite. Graphite is very porous and could not completely be deoxygenated. TorA-FDH was immobilized onto a glassy carbon electrode by adsorption and trapped under a permselective membrane. The biosensor was evaluated using amperometry. When the electrode is polarized at -0.8 V a rapid evolution of a catalytic reduction current is seen after TMAO addition to a buffer containing MV as soluble mediator (Fig. 2). Fig. 2 shows the typical steady state response curve and the schematic of the reaction sequence of the biosensor. A small peak appears immediately after the injection of TMAO, which vanishes within seconds and can be explained by disturbances from the sample injection, while a large stationary reduction current evolves. TMAO is reduced to TMA with concomitant oxidation of the enzyme. The reducing equivalents are delivered by MV +• , which is regenerated at the electrode. As a result, a catalytic current is generated proportional to TMAO concentration. No significant reduction current appeared when the enzyme was not immobilized on the electrode surface. or 50 µМ) depending on the needed sensitivity for the detection of DMSO (Abo et al., 2003).

To guarantee no mediator limitation for all further measurements of TMAO, we used 250 µM of MV.

The amount of enzyme used for immobilization was additionally varied. Increase of the enzyme amount (and thus the enzyme activity) led to a growth of sensor sensitivity until a maximum indicating that the sensor response changed from kinetic to diffusion limitation.

Fig. 3 represents the results of the loading test. Remarkably, the maximum response for TMAO is already reached at very low amount of TorA-FDH (7,07 pmol/cm 2 ) (Fig. 3). Due to the higher enzymatic activity of TorA-FDH, its loading is much lower compared to other described enzyme electrodes in which an enzyme is adsorbed on electrode surfaces (Schulz et al., 2014;Tavahodi et al., 2017). However, a tenfold reduction of the applied amount resulted in a clear drop of response. In order to have sufficient excess of enzyme and the highest response for further studies, we applied 5.9 nmol/cm 2 . Enzyme excess typically secures stability and longer measuring range.

Comparison between TorA-FDH and TorA-WT

In our previous work, we showed that the current increase at 3 mM TMAO in the presence of 1 mM MV was nearly the same for TorA-FDH and TorA-WT (81.99±8.53 and 83.16±3.5 µA, respectively), indicating that TorA-FDH and TorA-WT are comparably active (Kaufmann et al., 2018). Here, we compared the response of the immobilized TorA-WT and TorA-FDH under the newly defined conditions. From Fig. 4, it is obvious that now the enzyme response is not similar. In the presence of 100 µM of TMAO, TorA-WT is less efficient than TorA-FDH (323±81 nA vs. 440±26 nA, respectively). The current response for the TorA WTmodified electrode for 2 µM TMAO is also visibly decreased to 19.3±15 nA, which is almost half of that of the TorA-FDH modified electrode (28.3±1 nA). Furthermore, the response of the TorA WT sensor is less reproducible between sensor preparations. The larger variation between the sensors can be explained by differences in the sensitivity to oxygen since the TorA WT was expressed and purified under anaerobic conditions. It was already reported that a contact of the WT enzyme with oxygen can damage the cofactor and as a result lower the activity of the enzyme (Kaufmann et al., 2018).

Since the TorA-FDH variant seems to be less susceptible to oxidative damage compared to the WT enzyme it was selected for the construction of the biosensor for TMAO detection.

Biosensor performance

The biosensor was thus constructed with TorA-FDH immobilized on the electrode surface (5.9 nmol/cm 2 ) and addition of MV (250 µM). As shown by the above results, this biosensor can be used even in the presence of oxygen since the chimeric TorA-FDH enzyme remains stable. The performances of the biosensor were then tested. µM for both man and woman (Kühn et al., 2017;Wang et al., 2014). Higher values of around 100 µM TMAO were reported mostly for patients with chronic kidney disease but also associated with other disease states (Mafra et al., 2014;Stubbs et al., 2016;Zhu et al., 2016).

As can be seen from the graph (Fig 5A) the linear range is in the total range of our interest. To validate the oxygen removal system and confirm our results, the same measurement was performed except that additional N 2 purging was applied before and during the measurement (Fig 5A,squares). Thus, the measuring buffer containing GOx, Cat and glucose was purged with N 2 prior to the measurement for 30 min and then gently flushed continuously over the liquid phase during the whole measurement cycle. It is clear from the graph depicting the current versus the applied TMAO concentration that there is no difference in the performance between the sensor responses and thus, the O 2 removal capacity of the system is optimal and no residual oxygen interferes during the measurements.

Further, we also tested the reproducibility and stability of the biosensor. No obvious decrease of response was noted after 20 repetitive measurements over a period of 6 hours. The relative standard deviation (RSD) for these measurements is 8.8 %. Between each measurement the measuring cell was washed thoroughly with the measuring buffer. The results (SI Fig 4) indicate that the biosensor is stable for prolonged measurements during one day. After storage of the biosensor in 75 mM Tris buffer (currently used for enzyme storage in solution) at 4 °C for one week, the signal response dropped only by ≈ 13 % (12.67±4 %; SI Fig 5). Since the electrode is covered with a dialysis membrane which prevents leaching of the enzyme from the electrode surface, we ascribe this loss of the initial response to the enzyme inactivation.

Biosensor performance in serum

A potential application of the biosensor is the determination of TMAO in serum samples. indicate that the sensitivity is not markedly influenced when using real samples.

Conclusion

Here, we report on the development of the first enzyme-based TMAO biosensor involving a chimeric TMAO reductase from E.coli. Such design of recognition molecules represents a generic approach for new biosensors. Our results show that the biosensor made up with the chimeric enzyme has better characteristics than that with the wild type enzyme. The sensitivity and linear range of operation of the reported biosensor is in the desirable range for addressing the values of TMAO in blood of healthy people. The short-term usage stability is quite good. The sensor can operate over prolonged daily measurements. The serum measurements show that a detection of TMAO in human serum can be performed without interferences from the complex serum matrix. Furthermore, the incorporated O 2 removal system, consisting of GOx and Cat allows for performing measurements under ambient air conditions without intervening in the detection of TMAO and even high negative potentials can be applied without the risk of having overlapping signals from the O 2 reduction. Given the increased abundance of data for the involvement of TMAO in humans as a potential biomarker for several disease states, a fast and simple sensor for TMAO detection is preferred.

  5 mm was cleaned by polishing with emery paper with two different grit sizes (200 and 1500) and then ultrasonicated in Milli-Q water for 20 s and dried under N 2 stream. The electrode modification was carried out based on a published procedure(Aguey-Zinsou et al., 2002). Briefly the cleaned electrode was modified with a mixture of 1:1 volume ratio of 2 mM DDAB and 265 µM TorA-FDH. A 6 µL of this mixture was drop casted onto the electrode surface and left to dry in a desiccator at 4 °C for 15-20 min. The modified electrode was then transferred into the anaerobic glovebox and used for the voltammetric experiments. The same procedure was applied for the mediated and direct electrochemistry.2.3.2. BiosensorA glassy carbon electrode was cleaned with two different grain size Al 2 O 3 , 1 µm and 0.3 µm respectively, then ultrasonicated in Milli-Q water for 10 min, washed thoroughly and dried under N 2 stream. The electrode modification was performed by drop casting 5 µL of enzyme (TorA-FDH or TorA-WT) and drying in a desiccator at 4 °C for 30 min. Afterwards the electrode was covered with a dialysis membrane and fastened in the cell with an O-ring assembly. All biosensor measurements were performed under standard lab condition in air and at room temperature.

  Fig 1A is depicting the CV of the direct bioelectrocatalysis of TMAO by the immobilized TorA-FDH. After addition of 3 mM TMAO as substrate (black trace) a clear evolution of a biocatalytic reduction current can be seen. From the first derivative of the reduction part of the bioelectrocatalytic curve the catalytic potential Ecat = -0.56 V is determined (SI Fig 1). The catalytic potential (Ecat) is described as the maximum of the first derivative (dI/dE) or the inflection point where the bioelectrocatalytic current plateaus on both sides

Figure 1

 1 Figure1. A) Direct and B) mediated bioelectrocatalysis of TMAO by immobilized TorA-FDH. CV of the immobilized TorA-FDH electrode before (grey trace) and after (black trace) addition of 3 mM TMAO, recorded A) without and B) in the presence of 1 mM methyl viologen. Please note the different current ranges. Other conditions: Anaerobic, potassium phosphate buffer 10 mM pH 6.5; scan rate: 5 mV/s.

Figure 2 .

 2 Figure2. Amperometric curve of mediated reduction of 100 µM TMAO by immobilized TorA-FDH (left). On the right is a scheme representing the immobilized enzyme and the reaction with TMAO and the recycling of methyl viologen on the electrode surface. Potassium phosphate buffer 100 mM pH 6.5; Eappl -0.8 V; 8 U GOx, 200 U Cat and 50 mM glucose; 250 µM methyl viologen

Figure 3 .

 3 Figure 3. Enzyme loading dependence. Graph of the measured amperometric current versus the electrode applied TorA-FDH concentration. Potassium phosphate buffer 100 mM pH 6.5; TMAO 100 µM; Methyl viologen 250 µM; GOx 8 U, Cat 200 U and glucose 50 mM. (n=3 electrodes)

Figure 4 .

 4 Figure 4. Comparison between the activity of TorA-FDH and TorA-WT. Potassium phosphate buffer 100 mM pH 6.5; TMAO 100 µM; Methyl viologen 250 µM; GOx 8 U, Cat 200 U and glucose 50 mM; Eappl -0.8 V (n=3 electrodes).

Fig

  Fig 5A shows the dependence of the amperometric current on TMAO concentration. The useful measuring range is from 2 to 110 µM TMAO but the range of linearity is much longer (~10 mM TMAO) (SI Fig 3). The response time is 16±2 s and the sensitivity is 14.16 nA/µM TMAO with a limit of detection of LOD = 2.96 nM (S/N n = 3). The reported values of TMAO concentrations in the human blood plasma of a healthy person are between 2.25 -5.79

Figure 5 .

 5 Figure 5. Graph of the current response versus the concentration of TMAO A) N 2 purging additional on the O 2 removal system (squares) and without purging (triangles). B) in undiluted serum (triangles) and in buffer solution (squares). Potassium phosphate buffer 100 mM pH 6.5; Range of TMAO 2-110 µM; Methyl viologen 250 µM; GOx 8 U, Cat 200 U and glucose 50 mM, (n=3 electrodes)

  Therefore, recovery of TMAO in serum was tested and also the effect of serum on the sensitivity of the TMAO biosensor. An important point for the performance of this biosensor is the very small influence of the serum on the signal produced from the catalysis of TMAO by the immobilized TMAO reductase. In a series of experiments, we tested this aspect by injecting diluted serum spiked with 20 µM TMAO. The response was only 7 % lower for the spiked human serum sample compared to the same concentration of TMAO in buffer solution (67±0.5 nA vs. 72±1 nA, respectively)(SI Fig 6). This indicates that there are no substances in serum that can significantly interfere during the detection of TMAO by the immobilized TorA-FDH which is highly specific for TMAO.The next measurement was performed in undiluted serum containing additionally the O 2 removing system. After equilibration of the sensor the current generated upon consecutive additions of TMAO at different concentrations was evaluated. As can be seen from Fig5b(black squares) the linearity and sensitivity in the range between 2-110 µM are nearly the same compared to measurement in pure buffer solution (red circles) where the sensitivity in buffer solution is 14.16 nA/µM and in undiluted serum solution 13.75 nA/µM TMAO. This shows that the biosensor can perform the measurements without any interferences or need for serum dilution for detecting the TMAO in a real time serum sample. Altogether these results

Acknowledgment

The financial support by the Deutsche Forschungsgemeinschaft (DFG) for funding the Unicat cluster of excellence (EXC 314) is gratefully acknowledged. We would like to thank Kim Tiedemann for providing a sample of TorA-WT.

Supplementary Material

Click here to download Supplementary Material: Mitrova Supporting Information.pdf


Paul Kaufmann

Chantal Iobbi-Nivol

Silke Leimkühler

Protocols for the insertion of the bis-molybdopterin guanine dinucleotide cofactor into apo-molybdoenzymes from the DMSO reductase family

Molybdoenzymes are ubiquitous, and play important roles in all kingdoms of life. The enzymes' cofactors comprise the metal molybdenum, a special organic ligand system called molybdopterin (MPT), additional small ligands like water, hydroxide, oxo-, sulfido-or selenido-functions and, in some enzymes, a coordination to the peptide chain of the protein via an amino acid ligand (e.g. serine, aspartate, cysteine or selenosysteine). The so-called molybdenum cofactor (Moco) is deeply buried in the protein at the end of a narrow funnel giving access only to the substrate. In 1974 an assay was developed by Nason and coworkers using the pleotrophic Neurospora crassa mutant nit-1 for the reconstitution of molybdoenzyme activities from crude extracts. These studies lead to the understanding that Moco is the common element in all molybdoenzymes from different organisms. The assay has been further developed since using specific molybdenum enzymes as source of Moco for the reconstitution of diverse purified apo-molybdoenzymes. Alternatively, the molybdenum cofactor can be synthesized in vitro from stable intermediates and can be inserted into apo-molybdoenzymes by the aid of specific Moco-binding chaperones. A general working protocol is described here for the insertion of the bis-molybdopterin guanine dunucleotide cofactor (bis-MGD) into its target molybdoenzyme using the example of Escherichia coli TMAO reductase.

Introduction

Molybdenum is an essential trace element for bacteria, archaea and eukaryotes. In the cell, molybdenum is coordinated to the dithiolene group of the molybdenum cofactor (Moco) in almost all organisms [START_REF] Hille | The mononuclear molybdenum enzymes[END_REF]. Only bacteria which are able to fix nitrogen coordinate molybdenum additionally in the iron molybdenum cofactor (FeMoco) which is exclusively found in nitrogenase [START_REF] Hu | Biosynthesis of the iron-molybdenum cofactor of nitrogenase[END_REF]. In contrast, more than 60 different enzymes binding the Moco were identified in the past [START_REF] Leimkuhler | Bacterial molybdoenzymes: old enzymes for new purposes[END_REF]. The vast majority of molybdoenzymes is found in bacteria and achaea while in animals and plants only four or five different enzymes, respectively, are present [START_REF] Schwarz | Molybdenum cofactors, enzymes and pathways[END_REF]. The different Moco-containing enzymes are categorized on the basis of their molybdenum ligandation sphere, dividing them into three different enzyme families [START_REF] Hille | The mononuclear molybdenum enzymes[END_REF]: the xanthine oxidase (XO) family, the sulfite oxidase (SO) family and the dimethylsulfoxide (DMSO) reductase family (Figure 1). Enzymes of the XO family contain in general a Moco with a MPT-Mo VI OS(O -) ligand sphere, however, in bacteria the cofactor-core can also be present as molybdopterin cytosine dinucleotide cofactor (MCD) variant. Enzymes of the SO family contain a Moco with a MPT-Mo VI O2Cys core, while enzymes of the DMSO reductase family comprise a Moco with a MGD2-Mo VI XY core with X being either a sulfido-, selenidoor oxo-ligand and Y either being a hydroxo or amino acid ligand (Ser, Cys, Sec, or Asp). While eukaryotes produce only enzymes belonging to the SO and XO families, enzymes of all three families are present in prokaryotes with enzymes of the DMSO reductase family being the predominant ones. Nason et al. provided in 1974 the first biochemical evidence for a common cofactor to all Moco-containing enzymes, which was removed as a low molecular weight fraction from denatured Mo-enzymes of mammalian, plant or bacterial origin [START_REF] Nason | Evidence for a molybdenum cofactor common to all molybdenum enzymes based on the in vitro assembly of assimilatory NADPH-nitrate reductase using the Neurospora mutant nit-1[END_REF]. After separation, the protein-free Moco fraction was subsequently incorporated into a cofactor-free apo-nitrate reductase from eukaryotes (nit-1 extract), thereby activating the enzyme. The totality of these studies led to the understanding that with the exception of nitrogenase, Moco is the common element in all molybdoenzymes from different organisms. In addition to demonstrating the universality of Moco, the nit-1 extract assays also demonstrated that Moco is very labile with a lifetime of only a few minutes after release from molybdoenzymes, making chemical characterization of active Moco difficult. Therefore, structural characterization of Moco was achieved through the analysis of stable degradation products [START_REF] Leimkühler | The History of the Discovery of the Molybdenum Cofactor and Novel Aspects of its Biosynthesis in Bacteria[END_REF]. The chemical nature of Moco has been determined by Rajagopalan and coworkers in 1982 which revealed a reduced pterin with an unusual 6-alkyl side chain consisting of four carbons, a terminal phosphate ester and a unique dithiolene group critical for metal ligation [START_REF] Johnson | Structural and metabolic relationship between the molybdenum cofactor and urothione[END_REF]. Later crystal structures of Mo-enzymes have demonstrated the existence of a third pyrano ring between the OH-group at C3' of the side chain and the pterin C7 atom [START_REF] Schindelin | Crystal structure of DMSO reductase: redox-linked changes in molybdopterin coordination[END_REF][START_REF] Chan | Structure of a hyperthermophilic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase[END_REF][START_REF] Romão | The crystal structure of xnthine oxidase related aldehyde oxidoreductase[END_REF]. Once the pyrano ring is closed, a fully reduced hydrogenated pterin is formed. Because of the unique nature of the pterin in Moco, the metal-free form of the cofactor is called molybdopterin or metal-binding pterin (MPT), the latter reflects the fact that not only Mo but also W can be coordinated by this pterin scaffold.

The biosynthesis of Moco has been revealed to be highly conserved in all organisms, which can be divided into three general steps, according to the stable biosynthetic intermediates that can be isolated (14) (Figure 2): 1. the synthesis of cyclic pyranopterin monophosphate (cPMP) 2. conversion of cPMP into MPT by introduction of two sulfur atoms 3. insertion of molybdate to form Moco

In prokaryotes, a fourth step involves the further modification of Moco by the addition of nucleotide monophosphates to the phosphate group of MPT, forming the bismolybdopterin-guanine dinucleotide (bis-MGD) or MCD cofactor variants of the DMSO reductase or XO families, respectively [START_REF] Mendel | The biosynthesis of the molybdenum cofactors[END_REF]. While Nason and coworkers studied the reconstitution of apo-nitrate reductase from N. crassa, which belongs to the SO family of molybdoenzymes binding Mo-MPT ( 8), the first in vitro reconstitution of a bacterial enzyme of the DMSO reductase family with bis-MGD was reported in 1987 for nitrate reductase from Escherichia coli and was further optimized in the following decades showing that the activity of MobA is required for the assembly of the bis-MGD cofactor of nitrate reductase [START_REF] Giordano | Involvement of a protein with molybdenum cofactor in the in vitro activation of nitrate reductase from a chlA mutant of Escherichia coli K12[END_REF].

Studies on the molybdoenzyme TMAO reductase (TorA) from E. coli showed, that MobA acts in conjunction with the specific chaperone TorD in bis-MGD insertion into apo-TorA [START_REF] Pommier | TorD, a cytoplasmic chaperone that interacts with the unfolded trimethylamine N-oxide reductase enzyme (TorA) in Escherichia coli[END_REF]. By binding to the core of the apo-molybdoenzyme partner TorA, TorD induces a conformational change of apo-TorA that becomes competent for Moco insertion [START_REF] Genest | TorD, an essential chaperone for TorA molybdoenzyme maturation at high temperature[END_REF]. Further, TorD plays a role also in the last step of the Moco maturation by interacting with the MobA protein involved in bis-MGD formation [START_REF] Genest | Dedicated metallochaperone connects apoenzyme and molybdenum cofactor biosynthesis components[END_REF]. Thus, the chaperone acts as a facilitator to insert the synthesized bis-MGD cofactor into the binding site of apo-TorA [START_REF] Genest | Multiple roles of TorD-like chaperones in the biogenesis of molybdoenzymes[END_REF]. After bis-MGD insertion into apo-TorA, final folding of TorA is accomplished by the release of TorD from the complex. TorD-like proteins accordingly were considered to be systemspecific chaperones dedicated to the respective molybdoenzyme. In total, studies on the biosynthesis of Moco and the requirement of specific chaperones for the insertion of the various Moco derivatives after their synthesis suggested that Moco is a labile molecule that does not exist in a protein-free form in the cell [START_REF] Leimkühler | The History of the Discovery of the Molybdenum Cofactor and Novel Aspects of its Biosynthesis in Bacteria[END_REF]. Therefore, it was very surprizing that recent studies demonstrated that the complex bis-MGD cofactor can be extracted from the protein and reinserted into an apomolybdoenzyme without the help of a chaperone (to be accepted). The investigations showed that under anoxic conditions bis-MGD is stable for 90 min in its protein-free form. Further, extracted bis-MGD can be inserted into an apo-molybdoenzyme without the help of a chaperone which facilitates folding after insertion. Since the reconstitution of the N. crassa nit-1 extract with molybdenum cofactors from the SO and XO family of molybdoenzymes have been described in detail, we are focusing on the reconstitution of isolated molybdoenzymes from the DMSO reductase family with bis-MGD cofactors from cellular extracts, synthesized in vitro using MobA and TorD or direct insertion of intact bis-MGD by using apo-TorA. The major challenge for the insertion of bis-MGD into apo-molybdoenzymes lies in the fact that bis-MGD is labile towards O2, thus, all reconstitution steps must be performed under strictly anaerobic conditions.

Materials

Purified proteins required

A. Apo-TorA is expressed in a E. coli strain incapable of Moco biosynthesis (e.g. RK5200, ∆moaA [START_REF] Stewart | Nitrate reductase in Escherichia coli K-12: involvement of chlC, chlE, and chlG loci[END_REF][START_REF] Ilbert | Involvement of a mate chaperone (TorD) in the maturation pathway of molybdoenzyme TorA[END_REF]. A His6-tagged variant of TorA can be used to ensure simple purification [START_REF] Ilbert | Involvement of a mate chaperone (TorD) in the maturation pathway of molybdoenzyme TorA[END_REF]. The purified protein is stored in 100 mM Tris-HCl, pH 7.2 at -80°C until usage. The concentration of the protein should be at least 200 µM. A. The E. coli MobA protein is expressed as described previously [START_REF] Lake | The crystal structure of the Escherichia coli MobA protein provides insight into molybdopterin guanine dinucleotide biosynthesis[END_REF] as a His6-tagged variant and is stored in 100 mM Tris-HCl, pH 7.2 at -80°C until usage. The concentration of the MobA protein should be at least 100 µM B. The E. coli TorD protein is expressed as a His6-tagged variant and is stored in 100 mM Tris-HCl, pH 7.2 at -80°C until usage [START_REF] Ilbert | Involvement of a mate chaperone (TorD) in the maturation pathway of molybdoenzyme TorA[END_REF]. The concentration of the TorD protein should be at least 100 µM C. The protein used as bis-MGD source should be in a concentration of at least 300 µM in e.g. 100 mM Tris-HCl, pH 7.2 (or a buffer in which the protein is stable).

Rhodobacter capsulatus formate dehydrogenase (RcFDH) turned out to be an excellent bis-MGD donor protein which is used for the description of the method here. RcFDH is expressed as described previously as a His6-tagged protein and stored in 75 mM phosphate buffer, pH 7.2 at -80°C until usage [START_REF] Hartmann | The oxygen-tolerant and NAD-dependent formate dehydrogenase from Rhodobacter capsulatus is able to catalyze the reduction of CO to formate[END_REF]. The protein concentration should be around 600 µM, since the protein generally is only 50% loaded with bis-MGD.

In vitro reconstitution of apo-TorA from soluble extracts

1. Anoxic phosphate buffer 20 mM, pH 7 2. Purified E. coli TorD protein in 20 mM phosphate buffer, pH 7 3. Purified E. coli apo-TorA in 20 mM phosphate buffer, pH 7 4. 40 mg/ml soluble extract of an E. coli strain with a deletion in the genes for TMAO reductase and DMSO reductase (e.g. LCB504), in 100µl in phosphate buffer, pH7 5. Heat-block for Eppendorf tubes, electrophoresis system, a centrifuge and N2.

Reconstitution of apo-TorA with in vitro synthesized bis-MGD

1. Anoxic 100 mM Tris-HCl, pH 7.2 2. Anoxic 50 mM GTP 3. Anoxic 50 mM MgCl2 4. Purified E. coli TorD protein in 100 mM Tris-HCl, pH 7.2 5. Purified E. coli MobA protein in 100 mM Tris-HCl, pH 7.2 6. Purified E. coli apo-TorA in 100 mM Tris-HCl, pH 7.2 7. 300 µM purified Moco-donor molybdoenzyme from the SO (e.g. human or chicken sulfite oxidase) or XO family (e.g. bovine XO purchased from sigma), in 100 mM Tris-HCl, pH 7.2 8. Heat-block for Eppendorf tubes, a centrifuge and an anaerobic chamber (Coy chamber or comparable) suitable for work under an oxygen-free atmosphere 2.3 Reconstitution of apo-TorA with bis-MGD isolated from a donor protein 1. Anoxic 100 mM Tris-HCl, pH 7.2 2. Purified bis-MGD containing enzyme in a concentration of 300 µM (e.g. RcFDH) 3. Heat-block for Eppendorf tubes, a centrifuge and an anaerobic chamber (Coy chamber or comparable) suitable for work under an oxygen-free atmosphere

Methods

Scheme 1

3.1

In vitro reconstitution of apo-TorA with soluble extracts O2 free atmosphere can be obtained by bubbling N2 gas in Eppendorf tubes containing E. coli extracts or purified protein. However, the yield of reconstitution is lower as compared to using an anaerobic chamber. 1. E. coli cells from a strain LCB 440 (18) containing a deletion of the tor and dms operons grown anaerobically until the late exponential phase at 37°C 2. After centrifugation, the cells are resuspended in 20 mM phosphate buffer, pH 7 to 5 ml per g of cells 3. Cell lysis by French press or sonication, centrifugation for 20 min at 15,000 rpm 4. Discard the pellet, the supernatant is centrifuged in an ultracentrifuge for 90 min 45,000 rpm 5. Adjust the protein concentration to 30 -40 mg/ml with 20 mM phosphate buffer, pH 7 6. Keep the supernatant at 4 °C under nitrogen atmosphere 7. Mix in an Eppendorf tube:

-0.5 µM apo-TorA, -100 µl soluble extract (as bis-MGD source) -2.3 µM TorD 8. Adjust to a final volume of 150 µl with 20 mM phosphate buffer, pH 7 9. Keep for 2 hours at 37 °C under nitrogen atmosphere 10. The efficiency of the reconstitution can be checked by measuring the TorA activity in a spectrophotometric assay using benzyl viologen as electron donor

Reconstitution of apo-TorA in native gels

1. Mix in an Eppendorf tube: -1.5 µM apo-TorA -26.6 µM TorD 2. Adjust the final volume to 20 µl with 20 mM phosphate buffer, pH 7 3. Incubate for 1.5 hours at 37 °C, under nitrogen atmosphere Native PAGE: -Add 10 µl of non-denaturing loading buffer (200 mM Tris-HCl, pH 8.8, 5 mM EDTA, 1M sucrose, 0.1% bromophenol blue), do not boil the samples -Separate proteins on native 10% polyacrylamide gel -After separation, the polyacrylamide gel is incubated for 60 min at 37°C under nitrogen atmosphere, in 20 ml phosphate buffer containing 1 ml of bis-MGD source (1 ml soluble extract with a protein concentration of 40 mg/ml, preparation described above) -Transfer the polyacrylamide gel into 100 mM phosphate buffer, pH 6.5, containing 2 mM methyl viologen and 50 mM Na-dithionite in an anoxic atmosphere -When the gel turns dark blue, add 2 mM TMAO; active TorA will turn white in the gel, corresponding to the oxidation of reduced methyl viologen

In vitro reconstritution of apo-TorA with direct bis-MGD cofactor sources

In vitro reconstitution of apo-TMAO reductase with in vitro synthesized bis-MGD requires strict anaerobic conditions since Moco and its final product bis-MGD are highly sensitive to oxygen. All buffers, chemicals and proteins used need to be oxygen-free. We describe the preparation of samples i.e. in a Coy Chamber under a Nitrogen/Hydrogen atmosphere (95%/5%).

In vitro reconstitution of apo-TorA with in vitro synthesized bis-MGD using

MobA and TorD 1. Anoxic 100 mM Tris-HCl, pH 7.2 buffer is prepared at least one day before the reconstitution procedure. The buffer is sterile filtered and degassed for at least 1 hour followed by incubation in the Coy chamber for at least 12 hours to remove all oxygen. 2. For enzymatic hydrolysis of GTP magnesium chloride is required. A 50 mM MgCl2 solution is prepared, degassed as described in step 1 and incubated in the Coy chamber for at least 12 hours to remove residual oxygen. 3. On the day of reconstitution, 200 µM apo-TorA, 100 µM MobA protein in addition to the Moco donor protein in a concentration of 300 µM are incubated in the Coy chamber for at least 2 hours at 4 °C. In 2 ml Eppendorf tubes, GTP powder for a 50 mM stock solution is transferred to the Coy Chamber and incubated for at least 2 hours at 4 °C. 4. Extraction of Moco requires heat-denaturation of the donor protein. The donor protein needs to be diluted to 30 µM with the anoxic 100 mM Tris-HCl buffer, pH 7.2 in a total volume of 400 µl. The denaturation of the protein is carried out at 95°C for 4 minutes followed by centrifugation of the samples at 13,000 g for a minimum of 90 seconds. The supernatant is removed carefully and should not be stored for longer than 30 minutes under anoxic conditions. Long-term storage of isolated Moco is not recommended and will decrease the reconstitution efficiency. 5. The in vitro biosynthesis and simultaneous incorporation of bis-MGD into apo-TorA is started by mixing of 200 µl Moco-containing supernatant with 6 µl 50 mM GTP and 6 µl 50 mM MgCl2 (final concentration = 1 mM), 6 µl 100 µM MobA protein (2 µM final concentration), 6 µl 100 µM TorD protein and 20 µl 20 µM apo-TorA. Final volume of 300 µl is adjusted by addition of 62 µl of anoxic 100 mM Tris-HCl, pH 7.2. The reconstitution mixture needs to be incubated at 37°C for 7.5 hours under anoxic conditions. When higher end-concentrations of reconstituted TorA are required, the volume of the reaction can be generally scaled up, but it is important to keep the concentrations of all compounds as described above. An increase of the protein concentrations will result in a decreased reconstitution efficiency. 6. The reconstitution can be performed in the absence of TorD. The efficiency of the reconstitution is about 40% more effective when TorD protein is present in the assay. 7. After reconstitution the reconstituted active TMAO reductase needs to be purified from the reaction mixture to remove all low molecular weight compounds in addition to TorD and MobA. Gel filtration on a Superdex 200 or Superose 12 column, depending on the amount of sample, proved to be the method of choice.

In vitro reconstitution of apo-TMAO reductase with bis-MGD extracted from a DMSO-reductase family donor protein.

For reconstitution of apo-TorA with bis-MGD isolated from a bis-MGD containing donor protein, the MobA or TorD proteins are not required.

1. Anoxic 100 mM Tris-HCl, pH 7.2 buffer are prepared at least one day before the reconstitution procedure. After preparation, the buffer is sterile filtered and degassed for at least 1 hour followed by incubation in the Coy chamber for at least 12 hours to remove all oxygen. 2. On the day of reconstitution procedure 200 µM apo-TorA and 600 µM RcFDH are incubated in the Coy chamber for at least 2 hours at 4 °C. 3. Extraction of Moco requires heat-denaturation of the donor protein. The donor protein RcFDH is diluted to 30 µM with the anoxic 100 mM Tris-HCl buffer in a total volume of 400 µl (1:20). The denaturation of the protein is carried out at 95 °C for 4 minutes followed by centrifugation of the samples at 13,000 g for a minimum of 90 seconds. The supernatant is removed carefully and should not be stored for longer than 30 minutes under anoxic conditions. Long term storage of isolated Moco is not recommended and will decrease the reconstitution efficiency. The pellet of denatured RcFDH appears black, whereas the supernatant has a yellowish color. 4. The in vitro reconstitution of apo-TorA with bis-MGD is started by mixing 280 µl of the bis-MGD-containing supernatant with 20 µl of 20 µM apo-TorA. The reconstitution mixture is incubated at 37 °C for 7.5 hours under anoxic conditions. 5. After reconstitution, the activated TMAO reductase needs to be purified from the reaction mixture to remove all low molecular weight compounds. Gel filtration on a Superdex 200 or Superose 12 column, depending on the amount of sample, proved to be the method of choice.

Figure 1 :

 1 Figure 1: The families of molybdoenzymes. Shown are the different forms of the molybdenum cofactor (Moco, shown in the tri-oxo-form(6)) categorized into three different enzyme families are classified according to their coordination at the molybdenum atom: the xanthine oxidase, sulfite oxidase, and DMSO reductase families. The sulfite oxidase family is characterized by a MPT-Mo VI O2Cys ligand sphere. The xanthine oxidase family contains a MPT-Mo VI OS(O -) core (7). Here, the MPT core can be modified by an additional CMP nucleotide at the phosphate group, forming MCD. The DMSO reductase family contains a MGD2-Mo VI XY core with Y being either a sulfido, selenido-or oxo-ligand and X either being a hydroxo or amino acid ligand (Ser, Cys, Sec, or Asp).

Figure 2 :

 2 Figure 2: The biosynthesis of the molybdenum cofactors. Shown is a scheme of the biosynthetic pathway of Moco. The general pathway occurs via three conserved steps present in all organisms, the formation of cPMP, MPT and Mo-MPT, which are stable intermediates that can be isolated. Bacteria and Archaea contain a 4 th step of Moco modification in which Mo-MPT is further modified by the addition of nucleotides, either GMP or CMP. Additionally, Moco can be further modified by the replacement of one oxo-ligand by a sulfido-ligand, forming the sulfido-containing Moco present in the xanthine oxidase family of molybdoenzymes. After synthesis, the different forms of Moco are specifically inserted into the target enzymes of each family, either directly (SO family) or by the aid of molecular chaperones (examples are gives in blue).

Acknowledgements

The authors are supported by the Deutsche Forschungsgemeinschaft grants LE1171/6-2 and the Cluster of Excellence "Unicat" Exc314, coordinated by the TU Berlin (to S.L.), and the CNRS and AMU (to C. I.-N.)

6. TorD is not required for the insertion of bis-MGD into apo-TorA, therefore the reconstitution with extracted bis-MGD is described without TorD.

Notes

A. From preparation to preparation, and especially for TorA reconstituted with Moco from different Moco donors, the amount of incorporated bis-MGD into apo-TorA reductase will vary. To make all samples comparable, the relative FormA-GMP (stable and fluorescent oxidation product of MGD) has to be determined for each sample. Specific activities of reconstituted TorA can be correlated to FormA-GMP content per mg of enzyme to result in specific activities per unit of MGD (U/MGD) B. TMAO reductase of E. coli is highly stable in its holo form, whereas the apo form has moderate stability. Thus, removal of unreconstituted protein can be achieved by incubation of the reconstituted protein at 65 °C for 90 seconds with subsequent centrifugation at 13,000 g for 10 minutes. Apo-TorA will precipitate, whereas the holo-protein remains stable.