M Calvo-Dahlborg
email: monique.calvo-dahlborg@univ-rouen.fr

U Dahlborg
email: ulf.dahlborg@univ-rouen.fr.

J Cornide

S Mehraban
email: s.mehraban@swansea.ac.uk

Z Leong

T C C Hansen
email: hansen@ill.fr

R K K Wunderlich
email: rainer.wunderlich@uni-ulm.de.

R Goodall
email: r.goodall@sheffield.ac.uk

N P P Lavery
email: n.p.lavery@swansea.ac.uk

R Brown

S G R Brown

Structural investigation of the stability in

Keywords: High Entropy Alloys, multicomponent, structure, phases, calorimetry, e/a

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The term "high entropy alloys" (HEA) was introduced in 2004 to designate alloys consisting of 5 or more principal elements in equi-or near-equimolar amounts (5 to 35at%), being single-phased and having simple crystalline structures [START_REF] Yeh | Nanostructured high-entropy alloys with multiple principal elements: novel alloy design concepts and outcomes[END_REF]. The notion of high entropy refers to their high entropy of mixing. The last 15 years the alloys have been intensely investigated with respect to phase composition, microstructure, as well as thermal, magnetic and mechanical properties. However, it has also been shown that due to the unique combination of crystalline structures and microstructures several HEA can outnumber alloys today used in specific technological applications [START_REF] Gao | High-Entropy alloys: Fundamentals and applications[END_REF][START_REF] Zhang | Microstructures and properties of high-entropy alloys[END_REF][START_REF] Miracle | A critical review of high entropy alloys and related concepts[END_REF][START_REF] Miracle | Exploration and Development of High Entropy Alloys for Structural Applications[END_REF]. Thus, a new kind of metallurgy has emerged in that all elements have to be considered on an equal footing during the solidification process. This is different as compared to traditional technical alloys which usually consist of a leading ternary or binary system, such as for example Ti-Al, while the other elements are to be considered as minor additions. Thus, the designation, "multi-principal element alloys", is also used to define these kind of alloys [START_REF] Yeh | Nanostructured high-entropy alloys with multiple principal elements: novel alloy design concepts and outcomes[END_REF][START_REF] Senkov | Accelerated exploration of multiprincipal element alloys with solid solution phases[END_REF].

It is now well established that HEAs can be single-structured but not, or almost never, single-phased [START_REF] Dahlborg | Structure of some CoCrFeNi and CoCrFeNiPdx multicomponent HEA alloys by diffraction techniques[END_REF]. The identified structures belong in most cases to the cubic close-packed (fcc-type), the body centered cubic (bcc-type) and also hexagonal close packed (hcp-type) families of structures. It should though be noted that the identification of crystalline structures present in a many-phase alloy is a very complicated matter and its success depends on the resolution of the utilized experimental technique. Since their discovery the determination of the phase stability of HEAs after solidification has been a significant issue in many investigations.

Phase changes due to external fields (temperature, pressure, etc) have been found to have a profound influence on the microstructure and, thus, the mechanical and magnetic properties.

The design of alloys that contain phases with specific crystalline structures is accordingly of utmost importance in order to predict physical properties. For this purpose, a combination of parameters relevant in this connection have been considered including aspects of definition/terminology, phase formation, microstructure and phase stability, strengthening mechanisms, and high temperature properties, as well as the density and cost [START_REF] Gao | High-Entropy alloys: Fundamentals and applications[END_REF][START_REF] Zhang | Microstructures and properties of high-entropy alloys[END_REF][START_REF] Miracle | A critical review of high entropy alloys and related concepts[END_REF][START_REF] Miracle | Exploration and Development of High Entropy Alloys for Structural Applications[END_REF][START_REF] Yang | Prediction of high-entropy stabilized solid-solution in multi-component alloys[END_REF][START_REF] Tsai | High-entropy alloys: A critical review[END_REF][START_REF] Lu | An assessment on the future development of high-entropy alloys: Summary from a recent workshop[END_REF].

Recently a classification of HEAs was proposed using the average value of the electron concentration or number of itinerant electrons per atom (e/a) in the alloy and the average atomic radius calculated for a 12-atoms atomic neighbourhood [START_REF] Calvo-Dahlborg | Hume-Rothery for HEA classification and selforganizing map for phases and properties prediction[END_REF][START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF]. The importance of the number of itinerant electrons per atom to understand and predict the phases in solid solutions has been stressed as early as 1966 [START_REF] Barrett | Structure of metals[END_REF]. In [START_REF] Massalski | Comments Concerning Some Features of Phase Diagrams and Phase Transformations[END_REF] the use of e/a is discussed by Massalski for the determination of crystalline Hume-Rothery phases as opposed to an approach using the total number of valence electrons (VEC) (which includes the d electrons). By applying this approach for HEAs as solid solutions a classification and design methodology was proposed. From the variation of the average magnetic moment per atom and the hardness as a function of e/a and the 12 atoms radius, three domains could be identified in [START_REF] Calvo-Dahlborg | Hume-Rothery for HEA classification and selforganizing map for phases and properties prediction[END_REF] and [START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF]: one domain containing alloys with phases with close-packed cubic structures (below denoted fcc), a second one was found to contain alloys with phases of a mixed and complex structural type and a third one containing alloys with phases with body-centered cubic structures (below denoted bcc). The atomic radii are taken from Teatum [15] as these probably are the most useful when discussing metallic alloys. They are reported for a coordination number 12 atomic surrounding and were obtained from observed atomic distances in face-centered cubic and hexagonal close-packed structures. The first property used for classification of HEA's was the magnetization at saturation [START_REF] Calvo-Dahlborg | Hume-Rothery for HEA classification and selforganizing map for phases and properties prediction[END_REF][START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF]. The success of using high entropy alloys in most technological applications depends on their stability following different thermal exposures. The present work focusses on the influence of heat treatments on the stability of alloys and aims to investigate common effects in the three different domains after heat treatments. The alloys chosen for this investigation cover all domains according to the classification in [START_REF] Calvo-Dahlborg | Hume-Rothery for HEA classification and selforganizing map for phases and properties prediction[END_REF][START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF] and all contain Co, Fe and Ni as base components (abbreviated to CFN below). When Cr is also present in an alloy the abbreviation CCFN is used.

Material and methods

All alloys were prepared by arc melting of pure elements, with purities higher than 99.9 wt.%, under a Ti-gettered high-purity argon atmosphere [START_REF] Dahlborg | Structure of some CoCrFeNi and CoCrFeNiPdx multicomponent HEA alloys by diffraction techniques[END_REF]. The alloys were remelted several times in order to obtain homogeneity and to minimize oxide impurities. Cylindrical rods with diameter of 3mm were then prepared by copper-mold suction casting into a water-cooled Cu hearth. In order to investigate their thermal stability, the as-cast alloys were subjected to different heat treatments. Samples cut from the same ingots were annealed for 3 hours at 1100°C (T1), 700°C (T3) or 400°C (T2). In the following the temperature at which the samples had been heat treated will be referred to as T1, T2 and T3, the as-cast conditions as T0. The time needed to reach the required temperature was some few minutes. The heat-treated alloys were ice-water quenched in order to retain the structure. All subsequent measurements were performed at ambient temperature.

The crystalline structure of all samples was investigated by neutron diffraction (ND) on the D20 diffractometer at Institut Laue-Langevin (ILL), Grenoble (France). The wavelength of the incident neutrons was 1.12Å. The crystalline structure of the phases present in the alloys were determined by a refinement procedure using the DICVOL computer code [START_REF] Boultif | Powder pattern indexing with the dichotomy method[END_REF]. The values of the lattice constants will be presented in a separate paper. As neutrons are weakly interacting with matter the refinement gives a result valid for the entire irradiated volume.

The phase transformations occurring during heating up to different temperatures and during the subsequent cooling were studied for some selected alloys by Differential Scanning Calorimetry (DSC) with a Netzsch STA 449 C instrument under a flowing nitrogen protective atmosphere. The rate of temperature change was in all measurements 40Kmin -1 . It will be called "slow" in the following as opposition to "quench" for the water quench.

Results

Determination of the crystallographic structure of as-cast and annealed alloys by neutron diffraction

The structure of 12 selected alloys, 4 in each of the three domains that were defined in [START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF], will be discussed in this work. All have been investigated in as-cast condition as well as after two different heat treatments, namely T1 and T3. The measured diffraction patterns are presented in Fig. 1 to 3. For clarity the three curves for the different heat treatments have been shifted vertically and presented in the following order, from bottom to top: as-cast alloys (T0), after annealing for 3h at 1100°C (T1) and after annealing for 3h at 700°C (T3). When comparing the diffraction patterns, it is important to note that in the experiments the samples, all being ingots of different shapes and sizes, have been held in arbitrary orientations. Thus, the measured diffraction peak intensities are also affected by texture and they will therefore not necessarily give a representative estimation of the amount of a certain phase present. It should also be noted that all alloys have been produced and treated in the same way. Thus, no significant influence on the alloy phase composition due to a variation of elemental purities or on differences in cooling rates on diffraction peak intensities is expected. The structures identified from the diffraction patterns displayed in Figs 1 to 3 are listed in Table 2. A relative rough estimate of the amount of a particular structure in columns T0, T1 and T3 is given from their positions left to right, the one to the left being the main one. As the elemental composition of the different phases/structures has not been determined with enough accuracy only the type of crystalline structure is given. The question marks indicate the presence of a trace structure that could not be identified. The first column in Table 2 indicates the domain to which the alloy belongs according to the classification mentioned above [START_REF] Calvo-Dahlborg | Hume-Rothery for HEA classification and selforganizing map for phases and properties prediction[END_REF][START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF].

It can be seen that several alloys exhibit two phases having the same type of structure, for examples two simple fcc and one bcc in combination with a B2. The derived lattice constants of the different structures are presented in other works [START_REF] Dahlborg | Structure of some CoCrFeNi and CoCrFeNiPdx multicomponent HEA alloys by diffraction techniques[END_REF][START_REF] Calvo-Dahlborg | Structure and microstructural characterization of CoCrFeNiPd High Entropy Alloys[END_REF][START_REF] Calvo-Dahlborg | Interplay of electronic, structural and magnetic properties as driving feature of high entropy CoCrFeNiPd alloys[END_REF].

Table 2: List of the crystalline structures observed in alloys in as-cast condition (T0) as well as after heat treatments T1 and T3 defined in the text. The specified structures are the simple facecentered cubic (fcc), the simple body-centered cubic (bcc), the ordered body-centered cubic (B2), the Heusler (L21), and the hexagonal close-packed (hcp) structural type. The presence of unidentified structure is denoted by (?). The first column indicates the domain to which the alloy belongs according to the classification discussed in [START_REF] Calvo-Dahlborg | Hume-Rothery for HEA classification and selforganizing map for phases and properties prediction[END_REF][START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF]. All the alloys belonging to a specific domain have been found to have the type of structure in the as-cast state as predicted by the classification in [START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF]. Thus, in domain I alloys with fcc or hcp structures are found while the alloys in domain III contain structures of bcc type. The alloys in domain II form structures with both fcc and bcc type. The L21 phase is observed for a few alloy compositions and also after different heat treatments. It is a complex structure and contains atoms in both octahedral and tetrahedral interstitial positions with all atoms located on the sites of a body-centered cubic lattice.

On heat treatment it can be seen that the alloys of a particular domain behave similarly.

The investigated alloys of Domain I retain fcc and hcp phases after both T1 and T3 heat treatments (see Fig.

Calorimetric studies

In order to obtain further information on the thermal stability of the alloy structures differential scanning calorimetry (DSC) measurements were performed on one alloy from each Domain: CCFNPd from Domain I, CCFNAl from Domain II and CFNAl from Domain III. The heating/cooling rate was 40Kmin -1 for all measurements and all measurements were performed under continuous nitrogen gas flow. It should be noted that all studied alloys have melting points above 1400°C and that the solidification takes place during a slow cooling. The alloys were investigated according to different heat treatment schemes:

• A. Heating from room temperature (RT) to 1500°C on an as-cast sample previously annealed 3h at 1100°C and subsequently water-quenched.

• B. Heating from RT to 1500°C on an as-cast sample previously annealed 3h at 700°C and subsequently water-quenched.

• Ci (i=1,3). Heating/cooling of an as-cast sample according to the temperature variational scheme shown in Fig 4 . That is:

-C1: Heating of an as-cast sample from RT to 1100°C and subsequently cooled at the same rate (40K/min).

-C2: Heating of sample C1 from RT to 700°C and subsequently cooled at the same rate (40K/min).

-C3: Heating of sample C2 from RT to 1500°C. The curves are shifted vertically in the following order from bottom: Scheme A (black), scheme B (red), scheme C1 (green), scheme C2 (blue), scheme C3 (magenta). The vertical lines correspond approximately to the average temperature in the corresponding column in table 3.

Discussion

When comparing and interpreting the diffraction measurements in combination with the DSC studies it should be realized that the alloys have not been investigated under completely comparable circumstances by the two techniques. For experimental reasons the maximum temperature achievable in the neutron diffraction measurements was too low in order to duplicate the DSC measurements. Thus, all diffraction measurements were conveniently performed at ambient temperature on both the as-cast alloys as well as on the heat-treated ones, whereas the DSC measurements are performed during continuous temperature changes.

Consequently, some features seen in the measured DSC curves or in the diffraction patterns might not be detected by the other technique as the response to structural phase changes is different.

Domain I alloys

The diffraction patterns shown in Fig 1 and the reported phases identified in Table 2 show that the two annealing procedures T1 and T3 do not change the overall crystalline heat treatment implying the presence of a third structure. It was suggested in [START_REF] Dahlborg | Structure of some CoCrFeNi and CoCrFeNiPdx multicomponent HEA alloys by diffraction techniques[END_REF] that these were originating from a slightly distorted fcc structure (actually a monoclinic structure with a very similar lattice constant). This view is supported by DSC results discussed below.

The features observed in the DSC heat flow curves (Fig. 5a andb) at temperatures less than 1200 o C for CCFNPd alloy are summarized in Table 3. In order to substantiate the magnitude, the small ones are listed within parenthesis and the trace ones within double parenthesis. The melting temperature is found to be nearly the same in all the heating schemes and is found in the range 1276±4°C. The identified features are represented by vertical bars in Fig. 5a.

Table 3: Temperatures in °C at which features are identified in the DSC curves recorded for CCFNPd.

The as-cast CCFNPd alloy exhibits many structural transformations when heated to 1100 o C (scheme C1). Some of these are reversible and can be seen again during the second heating after heat treatments T1 and T3. The phase transformation occurring at the temperature Ta clearly takes place irrespective of the heat treatments of the alloy. It is very distinct after T1 and T3 but it is rather faint in the C3 heating. The corresponding phase has obviously grown during the 3h annealing time which shows the influence of both cooling rate annealing time on the alloy phase content. The DSC curves measured in the C sequence are very similar and show that the T1 and the T3 treatments when followed by slow cooling (40K/min) do not affect the alloy phase content significantly. Thus, all structural changes are reversible in this case. The DSC curves for as-cast CFNAl alloy (scheme C1) exhibit more features than for domain I and II alloys but the magnitude of these is considerably smaller. The T1 and T3 heat treatments followed by quench (A and B) seem to stabilize some of the phases. The transformation occurring at the temperature Tc might correspond to the Tb one at about 630 o C that was observed for the CCFNAl alloy (see Table 5). Some features disappear when the annealing is followed by a slow cooling (C3). Thus, the alloy is very sensitive to the rate of cooling.

It can be concluded that the crystalline structure of alloys classified to belong to domain III does not change during temperature treatments. All identified crystalline phases in this domain are of bcc, B2 or L21 type (Table 2).

The alloy phase content depends on the rate of temperature changes. The T1 and T3 heat treatments followed by quench (A and B) seem to stabilize some of the phases. Some features disappear when the annealing is followed by a slow cooling (C3). The slow cooling stabilizes only the Td transformation.

Domain II alloys

Some diffraction patterns measured for alloys that have been classified in [START_REF] Calvo-Dahlborg | Hume-Rothery for HEA classification and selforganizing map for phases and properties prediction[END_REF][START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF] From this domain the CCFNAl alloy was selected for DSC measurements according to temperature schemes mentioned above. The measured curves are shown in Fig. 7 and temperatures at which some specific features are observed are summarized in Table 5. The identified features are represented by vertical bars in Fig. 7. The onset of melting was for schemes A and C3 found to take place at 1350°C while after scheme B it was somewhat higher, at 1363°C. Two main transformations occur, one reversible at about 630 o C (Tb in Table 5) and one partly reversible at about 990 o C (Te in Table 5). The fcc structure observed in the diffraction pattern of this alloy after the T1 annealing and not after the T3 one can thus be correlated to Te feature in the DSC curve.

It can be concluded that the crystalline structure of alloys classified to belong to domain II change a lot during temperature treatments. This is obvious due to the presence in these alloys of mixed phases, e.g. metastable phases, intermetallics, sigma, etc. These phases naturally grow or transform to more stable phases under heat treatments. Thus, structural changes, both reversible and irreversible, might occur due to different heat treatments.

Conclusion

1) All the alloys belonging to a specific domain have been found to have the type of structure in the as-cast state as predicted by the classification in [START_REF] Calvo-Dahlborg | Hume-Rothery for HEA classification and selforganizing map for phases and properties prediction[END_REF][START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF]. Thus, in Domain I alloys with fcc or hcp phases are found while the alloys in Domain III contain phases of bcc type. The alloys in Domain II form phases with both fcc and bcc structure as well as other intermetallic phases.

2) Removing Cr from one alloy can drive the alloy from one Domain to another. A small change in the relative Al amount of an alloy can have the same effect. Both these effects are depending on the resulting values of e/a and r [START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF].

3) The investigated alloys of Domain I exhibit only one type of structure, whatever heat treatment and cooling rate has been used. The microstructure transforms from multi-to almost single-phase under homogenization at T=1100°C. A heat treatment at 700°C leads to additional phases depending on the cooling procedure after annealing.

Fig. 1 :

 1 Fig. 1: ND patterns of the investigated alloys of Domain I in (black) the as-cast (T0) and after heat treatment at (red) T1 and (blue) T3 condition. The alloy compositions are a) CoCrFeNiPd, b) CoCrFeNiRu, c) CoCrFeNiCu, and d) CoFeNiCu. The vertical bars correspond to diffraction peak positions of the two crystalline structures present in largest amounts in every alloy.

Fig. 2 :Fig. 3 :

 23 Fig. 2: ND patterns of the investigated alloys of Domain III in (black) the as-cast (T0) and after heat treatment at (red) T1 and (blue) T3 condition.The alloy compositions are a) CoFeNiAl, b) CoFeNiAlTi, c) CoCrFeNiAl1.2, and d) CoFe0.8NiAl1.2. The vertical bars correspond to diffraction peak positions of the two crystalline structures present in largest amounts in every alloy.

  1 a to d). Alloys of Domain III retain phases of bcc types (see Fig 2 a to d) and alloys of Domain II retain their mixed phase composition (see Fig. 3 a to d). Another result from Table 2 is that removing Cr from one alloy sometimes drives the alloy from one Domain to another (e.g. CCFNAlTi and CCFNAl in domain II, CFNAlTi and CFNAl in Domain III) and sometimes not (e.g. CCFNCu and CFNCu in Domain I). It is also interesting to note that a small change in the relative Al amount of an alloy can have the same effect (e.g. CCFNAl in Domain II and CCFNAl1.2 in Domain III). Both these effects depend on compared values of e/a and r [12].

Fig. 4 .

 4 Fig. 4. Temperature variational scheme applied during DSC measurements of as-cast alloys previously heated to 1100°C and subsequently cooled with a rate of 40K/min.

Fig. 5 :

 5 Fig. 5: (a) DSC heat flow curves and (b) their derivative measured during the five different temperature schemes for the CCFNPd alloy belonging to domain I.The curves are shifted vertically in the following order from bottom: Scheme A (black), scheme B (red), scheme C1 (green), scheme C2 (blue), scheme C3 (magenta). The vertical lines correspond approximately to the average temperature in the corresponding column in table 3.

Fig. 6 :

 6 Fig. 6: (a) DSC heat flow curves and (b) their derivative obtained for the five different temperature schemes for the CFNAl alloy belonging to domain III. The curves are shifted vertically in the following order from bottom: A (black), B (red), C (green), D (blue), E (magenta). The vertical lines correspond approximately to the average temperature in the corresponding column in table 4.

Fig. 7 :

 7 Fig. 7: (a) DSC heat flow curves and (b) their derivative obtained for the five different temperature schemes for the CCFNAl alloy belonging to domain II. The curves are shifted vertically in the following order from bottom: A (black), B (red), C (green), D (blue), E (magenta). The vertical lines correspond approximately to the average temperature in the corresponding column in table 5.

  structures of the alloys. Only two different structures are observed, namely fcc and hcp. The corresponding diffraction peak position are shown as vertical bars in Figs 1. The only exception is the CCFNPd alloy for which the measured pattern exhibits several extra peaks after the T3

  to belong to domain II are shown in Fig 2. All contain structures of both fcc and bcc type irrespective of heat treatments. Only diffraction peak positions corresponding to the two major structures are displayed as vertical bars. Thus, the clearly visible peaks that correspond to the B2 structure in the Figs 2a and2bare not explicitly shown. There does not seem to be a uniform behavior of the alloys in this domain on any of the heat treatments T1 and T3 in that for some new structures appear and for some not.

4 ) 5 ) 5 )

 455 Alloys of Domain II undergo more phase transformations during the different heat treatments than the investigated Domain I alloy. The alloys contain both fcc and bcc phases (being of mixed type) and are still of mixed type after heat treatment. Most of the phases present in as-cast samples remain after heat treatment at 1100 and 700°C indicating a pronounced structural instability of the alloy with regard to temperature variations. The relative strength of the different phase changes with the rate of cooling. Alloys from Domain III present more transformations in the as cast sample than for domain I and II alloys but these features are much smaller. The 1100 and 700°C heat treatments followed by quench seem to stabilize some of the phases. The alloy phase composition is very sensitive to the rate of cooling. The importance of rate of cooling after annealing at 1100 and 700°C influences the phase composition and their amount. This a very important result for the homogenization procedure often performed on HEAs, depending in the Domain where their compositions are locating them.

Table 1

 1 gives the acronyms and the nominal compositions of the investigated alloys.

	Acronym	Alloy nominal composition (at.%)
	CCFNPd	Co20Cr20Fe20Ni20Pd20
	CCFNRu	Co20Cr20Fe20Ni20Ru20
	CCFNCu	Co20Cr20Fe20Ni20Cu20
	CFNCu	Co25Fe25Ni25Pd25
	CCFNAl	Co20Cr20Fe20Ni20Al20
	CCFNAlCu	Co16.6Cr16.6Fe16.6Ni16.6Al16.6Cu16.6
	CFNTi	Co25Fe25Ni25Ti25
	CCFNAlTi	Co16.6Cr16.6Fe16.6Ni16.6Al16.6Ti16.6
	CFNAl	Co25Fe25Ni25Al25
	CFNAlTi	Co20Cr20Fe20Ni20Ti20
	CCFNAl1.2	Co19.2Cr19.2Fe19.2Ni19.2Al23.1
	CF0.8NAl1.2	Co25Fe20Ni25Al30

Table 4 :

 4 The diffraction measurements indicate that a new phase was created in the CCFNPd alloy after the T3 treatment (Fig1a). It might be conjectured that this corresponds to the transformation taking place at the temperature Tc. As the transformation probably is of diffusive nature it has been initiated but not completed during the three hours the alloy was kept at 700 o C. Temperature in o C at which features are identified in the DSC curves recorded for the CFNAl alloy.

	Measurement Ta	Tb	Tc	Td	Te	Tf	Tg	Th
	A	580	(717)	793			(1036)
	B	587		((793))		(991)	1057	1110
	C1	593	(727)	791	916	987	(1041)
	C2	(561)
	C3	(590) (720)	(797)	917	980	(1040)		1190

Table 5 :

 5 Temperature in °C at which features are identified in the DSC curves recorded for CCFNAl.

	Measurement Ta	Tb	Tc	Td	Te	Tf	Tg	Th
	A		627	776		988	1048	1110
	B	((506)) 629	757	831	988	((1071))		1165
	C1	(443)	638	754	(850) (991)	(1056)
	C2	((527)) 630
	C3		629	(764)	(858) 990	((1056))

It can be concluded from the shape of the DSC curves that the structure of this alloy is affected more significantly by temperature changes than the CCFNPd belonging to domain I.

Acknowledgements

The authors are thankful to Institute Laue-Langevin, Grenoble, France, for awarding beam-time on the D20 diffractometer at the ILL reactor. Thanks are due to N.J.E. Adkins, at The University of Birmingham (UK) for making the CoFeCu alloy investigated in this study. The present work has been carried out within the FP7 European project AccMet NMP4-LA-2011-263206 for. Some of the DSC measurements have been performed on installations founded by the COMET project at Swansea University.

It can be concluded that the crystalline structure of alloys classified to belong to domain I does not change during temperature treatments. All identified crystalline phases in this domain are of fcc and/or hcp type (Table 2). Structural changes, both reversible and irreversible, might occur due to different heat treatments but all phases remain of this type. However, the alloy phase content depends on the rate of temperature changes.

Domain III alloys

Some diffraction patterns measured on alloys that have been classified in [START_REF] Calvo-Dahlborg | Hume-Rothery for HEA classification and selforganizing map for phases and properties prediction[END_REF][START_REF] Calvo-Dahlborg | Influence of the electronic polymorphism of Ni on the classification and design of high entropy alloys[END_REF] 4. The identified features are represented by vertical bars. The general shapes of the curves are very different from the ones measured for the CCFNAl alloy (see Fig 7) indicating that the temperature behavior is sensitive to the Cr content. A very striking observation is that the melting takes place in two stages after scheme C3. The sum of areas of the two peaks is close to the areas of the corresponding peaks measured after schemes A and B. This suggests that the alloy consists of two phases after schemes C1 and C2. The temperatures at which the melting processes take place Tm1 and Tm2 are shown in table 4.


Kien Lam Ung

Laurent Kremer

Mickael Blaise
email: mickael.blaise@irim.cnrs.fr

M Abscessus Eis1

Mickaël Blaise

Structural analysis of the N -acetyltransferase Eis1 from Mycobacterium abscessus

Keywords: Mycobacterium abscessus, N-acetyltransferase, aminoglycosides, Eis, GCN5, X-ray structure

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

N-acetyltransferases are enzymes catalyzing the transfer of acyl groups from acyl-CoA to a wide panel of substrates, ranging from small molecules like spermine to large macromolecules, such as histones. Prokaryotic N-acetyltransferases belonging to the GCN5-related Nacetyltransferases family (GNAT) are capable to modify a large set of small substrates, including aminoglycoside antibiotics. They can exert pleiotropic effects, participating for instance in the biosynthesis of mycothiol, a mycobacterial antioxidant, or can modify bacterial peptides/proteins as well as host proteins in the case of pathogenic bacteria [START_REF] Favrot | Bacterial GCN5-Related N-Acetyltransferases: From Resistance to Regulation[END_REF] .

The Eis protein was initially discovered to confer enhanced survival of Mycobacterium smegmatis in macrophages, hence its designation as Enhanced Intracellular Survival 2 . Numerous studies have subsequently addressed the function of Eis from Mycobacterium tuberculosis (Eis Mtb ) emphasizing its capacity to modulate the host inflammatory response and autophagy [START_REF] Duan | Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3[END_REF][START_REF] Samuel | Expression, production and release of the Eis protein by Mycobacterium tuberculosis during infection of macrophages and its effect on cytokine secretion[END_REF][START_REF] Shin | Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling[END_REF] as well as its activity to acetylate various proteins, including the host histone H3 [START_REF] Duan | Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3[END_REF] and the mycobacterial nucleoid-associated protein HU [START_REF] Ghosh | Lysine acetylation of the Mycobacterium tuberculosis HU protein modulates its DNA binding and genome organization[END_REF] .

In M. tuberculosis clinical strains, mutations in the eis promoter resulted in the up-regulation of eis expression and resistance to kanamycin [START_REF] Zaunbrecher | Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis[END_REF] . The role of Eis Mtb in the modification of aminoglycosides has been the subject of deep biochemical and structural investigations, demonstrating that its belongs to the GNAT superfamily of proteins. Eis Mtb forms a homohexamer and can modify a wide range of aminoglycosides through mono-or poly-acetylations of their primary amine groups [START_REF] Green | Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria[END_REF][START_REF] Houghton | Chemical and structural insights into the regioversatility of the aminoglycoside acetyltransferase Eis[END_REF] .

Consistent with these findings, the Eis2 protein (Eis2 Mab ) has been shown to also play a key role in Mycobacterium abscessus, a non-tuberculous mycobacterium (NTM), recognized as an emerging human pathogen [START_REF] Johansen | Non-tuberculous mycobacteria and the rise of Mycobacterium abscessus[END_REF] . The M. abscessus complex comprises three subspecies, M. abscessus sensu stricto, M. abscessus subsp. bolletii and M. abscessus subsp. massiliense and has been associated with various clinical manifestations, including cutaneous, soft tissues, disseminated infections or lung infections. The pulmonary infections are particularly problematic to treat in the context of cystic fibrosis patients that are highly vulnerable to M. abscessus complex [START_REF] Roux | Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in france[END_REF][START_REF] Dedrick | Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus[END_REF] . M. abscessus is also recognized as one of the most antibiotic-resistant mycobacterial species, leaving clinicians with few therapeutic options [START_REF] Nessar | Mycobacterium abscessus: a new antibiotic nightmare[END_REF] . Standard drug regimens against M. abscessus lung diseases usually combine three antibiotic classes: an aminoglycoside (amikacin), a macrolide (clarithromycin) and a β-lactam (cefoxitin or imipenem) [START_REF] Strnad | Treatment of Mycobacterium abscessus Complex[END_REF] . However, M. abscessus is often refractory to these chemotherapies. The extreme resistance levels of this bacteria to most conventional antibiotics is notably well exemplified by the fact that M. abscessus is resistant to all first-line anti-tubercular drugs (isoniazid, rifampicin, pyrazinamide) [START_REF] Nessar | Mycobacterium abscessus: a new antibiotic nightmare[END_REF] .

In addition to drug target modification, resistance mechanisms in M. abscessus involve also intrinsic drug resistance, low permeability of the cell wall, induction of drug efflux pumps, absence or non-functional drug-activating enzymes which no longer convert pro-drugs into active metabolites.

Amikacin (AMK), a widely used antibiotic for the treatment of M. abscessus lung infection, is inactivated by the N-acetyltransferase Eis2 Mab [START_REF] Rominski | Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes[END_REF][START_REF] Ung | Crystal structure of the aminoglycosides N-acetyltransferase Eis2 from Mycobacterium abscessus[END_REF] . Biochemical and structural studies indicated that Eis2 Mab accommodates a wide range of aminoglycosides in its active site where they are modified by the addition of one or several acetyl groups on their primary amine function, thus impeding their interaction with the ribosome [START_REF] Ung | Crystal structure of the aminoglycosides N-acetyltransferase Eis2 from Mycobacterium abscessus[END_REF][START_REF] Green | Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria[END_REF] . Solving the high-resolution structure of Eis2 Mab proved that this protein accommodates amikacin in its active site where the drug undergoes acetylation as supported in enzymatic assays [START_REF] Ung | Crystal structure of the aminoglycosides N-acetyltransferase Eis2 from Mycobacterium abscessus[END_REF] . Importantly, the deletion of eis2 Mab increases the susceptibility of M. abscessus to aminoglycosides, thus supporting our findings modestly impacted the capacity of the mutant strain to infect macrophages and failed to modify the susceptibility to aminoglycosides as compared to an eis2 Mab mutant [START_REF] Dubois | Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages[END_REF] . This suggests that Eis1 Mab and Eis2 Mab share very limited overlapping functions [START_REF] Rominski | Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes[END_REF][START_REF] Dubois | Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages[END_REF] . The reasons for these differential biological functions between Eis1 Mab and Eis2 Mab remain obscure when considering the primary sequence identity (31%) between the two proteins. To fill this gap, and in order to provide the mechanistic basis explaining these differential aminoglycoside-modifying properties, a thorough structural and biochemical study of Eis1 Mab was undertaken. with SQ2_b_down. Identification of eis2 mutants was performed by PCR using this pair of primers:

Materials and Methods

1-

SQ1_a_up: 5'-gctgggatgacggcggatgggacgg-3' and SQ1_b_up: 5'-ggtggagcgaatagatcgcgtcatg-3'.

The positive clones were further confirmed by DNA sequencing of the genomic region surrounding the site of gene deletion.

For functional complementation of the eis1 Mab deletion mutant (Δeis1 Mab ), the genomic sequence of eis1 Mab was fused at its 5' end in frame with the sequence encoding the Streptag-II (italic letters), using the following primers (restriction sites in capital letters): fw_Eis1_261_NdeI The parental, Δeis1 and Δeis2 M. abscessus strains were transformed with either pMV261-Zeo, pMV261-Zeo-eis1 or pMV261-Zeo-eis2 and selected onto 7H10 agar supplemented with OADC and 50 µg.mL -1 Zeocin.

2-Amikacin susceptibility assessment.

The different strains were grown in Sauton's broth medium supplemented with 10%(v/v) OADC/ 0.025%(v/v) tyloxapol and 50 µg.mL -1 zeocin. The exponential bacterial cultures were pelleted and resuspended in PBS buffer. Five microliters of 10-fold serially diluted M. abscessus cultures were spotted on LB plates in the absence or presence of 4, 8 and 12 µg.mL -1 AMK. Plates were incubated at 37°C for 3 days.

3-Cloning, protein expression, and purification

The codon-optimized sequence (GenScript) encoding eis1 Mab was cloned into pET-41 between the KpnI and EcoRI restriction sites in frame with the N-terminal Glutathione S-transferase (GST) tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. washing the column with buffer E supplemented with 50 mM imidazole. The protein was concentrated using a 10 kDa cut-off centricon (Sartorius) and purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column (GE Healthcare) with buffer F (20 mM Tris-HCl pH 8, 0.2 M NaCl). The protein concentration was determined using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific) and the protein purity was estimated by

Coomassie blue staining. Eis2 Mab was expressed and purified, as described previously [START_REF] Ung | Crystal structure of the aminoglycosides N-acetyltransferase Eis2 from Mycobacterium abscessus[END_REF] .

4-Enzyme kinetic assays

Eis1 Mab and Eis2 Mab were freshly purified in buffer F. The CoA-SH product of the acetyltransferase reaction triggers the reduction of DTNB (5,5-dithio-bis(nitrobenzoic acid) to form TNB (2-thio-5-nitrobenzoic acid, extinction coefficient: 14150 M -1 .cm -1 ) with a peak of absorption at 412 nm. A master stock of KAN (containing 96% Kanamycin A and 4% KAN B), APR The reaction was initiated and measured immediately after combining both premixes to a final volume of 100 µL containing 0.25 µM enzyme, 0.5 mM ACO, 2 mM DTNB, and 5 mM of each substrate for assessment of the enzyme specificity. For kinetic constants determination one of the concentration either 5 mM TYR or 0.5 mM ACO was kept constant and the concentration of the other one varied. The reaction was followed at 25 o C and data points were recorded every 5 s for 90 s in a quartz cuvette and using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific). All absorbance values were normalized against a negative control where the substrate was absent and standard errors were calculated from three replicates. Nonlinear least-squares regression was used to determine the Michaelis-Menten equation (Prism software, GraphPad).

5-Assessment of protein expression by Western-Blot

Single colonies were inoculated in 100 mL of Sauton's broth supplemented with 10%(v/v) OADC/0.025%(v/v) tyloxapol and 50 µg.mL -1 zeocin and grown for 3 days at 37 o C, under agitation at 120 rpm. Bacteria were harvested by centrifugation and resuspended in buffer A, described above. After opening bacteria by bead-beating and sonication, samples were centrifuged at 20, 000 g, at 4 o C. The supernatant was collected, mixed with loading-blue buffer and separated on SDS-PAGE. Proteins were then transferred onto a nitrocellulose membrane (GE Healthcare) in transfer buffer (20 mM Tris, 150 mM Glycine, 20% (v/v) ethanol). The membrane was saturated by blocking buffer (PBS 1X, 5% (m/v) skimmed milk, 0.2% (v/v) Tween 20) followed by incubation with primary monoclonal Anti-Strep-tag antibodies produced in mouse (dilution 1/10000) (Sigma-Aldrich). Bands were revealed by polyclonal anti-mouse antibodies conjugated to the horseradish peroxidase (dilution 1/5000) (Sigma-Aldrich). Protein were detected by the luminol-based reaction (SuperSignal TM -Thermo Fisher Scientific) and imaged by a Chemidoc (Biorad).

6-Crystallization and x-ray data collection

7-Structure solving and refinement

The Eis1 Mab structure was solved by molecular replacement using the crystal structure of the Eis2 Mab monomer bound to ACO (PDB: 6RFT) as a search request and sharing 31 % primary sequence identity with Eis1 Mab . Molrep from the CCP4i package [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF] was used to performed molecular replacement. Six molecules were found in the asymmetric unit. After a first refinement step leading to R/Rfree of 0.46/0.5, Autobuild from the Phenix software suite [START_REF] Adams | PHENIX: a comprehensive Python-based system for macromolecular structure solution[END_REF] was further used to automatically improved the model building which led to R/Rfree: 0.32/0.41. The model was further manually rebuilt and refined using Coot [START_REF] Emsley | Features and development of Coot[END_REF] and the Phenix software suite. This led to a final model with refinement statistics shown in Table 1. The coordinates and structures factors have been deposited at the protein data bank under accession number: 6YCA.

Results

1-Overexpression of Eis1 Mab does not induce aminoglycoside resistance in M. abscessus

A previous study reported that the deletion of eis1 Mab , but not eis2 Mab , was associated with an unchanged susceptibility profile to aminoglycosides, leading the authors to conclude that only eis2 Mab participates in resistance to this class of antibiotics [START_REF] Rominski | Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes[END_REF] . However, as emphasized by these authors, additional studies are required to confirm or dismiss the implication of Eis1 Mab in aminoglycoside resistance, particularly by determining the antibiotic susceptibility pattern of a strain overexpressing Eis1 Mab . To address these two possibilities, eis1 Mab was cloned into the episomal pMV261-Zeo construct under the control of the strong hsp60 promoter, yielding pMV261-Zeo-eis1, to achieve high levels of protein expression. To easily monitor the expression of Eis1 Mab by Western blotting, the protein was expressed as a fusion protein harboring a Streptag-II in its N-terminus. Moreover, a Δeis1 Mab deletion mutant was generated in the smooth variant of M.

abscessus using a recently established recombineering procedure that allows the generation of unmarked gene deletion (Figure 1A and Figure 1B) [START_REF] Richard | Mutations in the MAB_2299c TetR Regulator Confer Cross-Resistance to Clofazimine and Bedaquiline in <em>Mycobacterium abscessus<[END_REF] . Both the parental and Δeis1 Mab strains were transformed with either the empty pMV261-Zeo or pMV261-Zeo-eis1. Zeocin was chosen as a selection marker as it does not belong to the aminoglycoside class of antibiotics and thus, the ZEO resistance cassette is very unlikely to affect the aminoglycoside susceptibility profile of the different strains tested. To include a control strain with increased susceptibility to aminoglycosides, we also generated a Δeis2 Mab deletion mutant known to be susceptible to AMK [START_REF] Rominski | Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes[END_REF] and complemented strain (Figure S1).

Probing bacterial lysates of three independent clones using anti-Strep tag antibodies testified that high expression levels of Eis1 Mab and Eis2 Mab were achieved in both the wild-type, Δeis1 Mab and Δeis2 Mab strains carrying pMV261-Zeo-eis1 or pMV261-Zeo-eis2 (Figure 1C and Figure S1).

Next, we tested whether overexpression of Eis1 Mab in both wild-type and Δeis1 Mab strains was associated with increased resistance to aminoglycosides on agar plates supplemented with increasing concentrations of APR, HYG or AMK. No major growth defect was noticed between the different strains plated on either drug as shown for AMK (Figure 1D). Conversely, the Δeis2 Mab mutant was hypersensitive to AMK as compare to all the other strains (Figure 1D). Overexpression of Eis2 Mab in the mutant rescued this effect and growth occurred even in the presence of 12 µg.mL -1

of AMK, the highest concentration tested (Figure 1D). These results corroborate previous findings suggesting that unlike Eis2 Mab , Eis1 Mab is not a primary determinant of resistance to aminoglycosides 17 .

2-Eis1 Mab is an active N-acetyltransferase but does not modify aminoglycosides

We next investigated the biochemical properties of Eis1 Mab . It was previously reported that recombinant Eis1 Mab tends to aggregate, rendering the purification procedure challenging [START_REF] Green | Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria[END_REF] .

Likewise, we were facing the same issues when Eis1 Mab was fused to small tags at its N-terminus, such as His-and S-tags. This tendency to aggregate may suggest a folding issue of the protein and, may explain, at least partly, the lack of activity of Eis1 Mab 8 . To circumvent this problem, we attempted to increase protein solubility by overexpressing Eis1 Mab fused to the Glutathione-Stransferase (GST) in its N-terminus. This greatly increased the solubility of the protein and to purify it via a three-step purification procedure. Eis1 Mab did not aggregate after removal of the GST-tag and, as long it was not too concentrated, a homogeneous and sharp peak could be observed on gelfiltration (Figure 2). The size-exclusion elution profile strongly suggests that Eis1 Mab behaves as a multimer in solution. A calibration curve allowed to calculate an apparent molecular weight of 198 kDa, similar to the one reported for its closest homologue Eis2 Mab 18 or to those of Eis from M.

tuberculosis or M. smegmatis [START_REF] Anand | Differential thermal stability, conformational stability and unfolding behavior of Eis proteins from Mycobacterium smegmatis and Mycobacterium tuberculosis[END_REF] . From this, it can be inferred that Eis1 Mab is a hexamer in solution.

The substrate specificity of Eis1 Mab was next assessed in vitro by enzymatic assay but no activity was detected towards a range of aminoglycoside antibiotics. However, an Nacetyltransferase activity was detected when using arylalkylamine substrates, such as TYR and HIS

(Figure 3A and3B). These two substrates are smaller compare with aminoglycosides (Figure 3A)

and possess primary amine groups. TYR and HIS are known to be acetylated by different Nacetyltransferases and are substrates of Eis from M. tuberculosis and M. smegmatis [START_REF] Pan | Eis, a novel family of arylalkylamine N-acetyltransferase (EC 2.3.1.87)[END_REF] . Interestingly,

Eis1

Mab was found to be more active towards TYR and HIS than Eis2 Mab (Figure 3B).

This prompted us to determine the kinetic constants of Eis1 Mab for TYR, which appears as the preferred substrate of the enzyme (Figure 3B). As could be expected for noncognate substrates the K M = 722 ± 21 µM and k cat = 11 ± 0.5 s -1 for TYR are suggestive of moderate activity. Eis1 Mab displays also a slightly lower affinity (K M= 144 ± 19 µM) for ACO as compared to Eis2 Mab (K M = 36.2 ± 2.9 µM) [START_REF] Ung | Crystal structure of the aminoglycosides N-acetyltransferase Eis2 from Mycobacterium abscessus[END_REF] and to Eis homologous proteins 8 which all display K M values < 50 µM (Figure 3C). Nonetheless, these in vitro data support the view that Eis1 Mab purified under our experimental procedure is an active N-acetyltransferase and, in contrast to Eis2 Mab, does not modify aminoglycosides. The smaller substrate preference of Eis1 Mab as compared to Eis2 Mab makes it a noticeable feature that distinguishes the two enzymes.

3-Overall crystal structure of Eis1 Mab

To get insights into the molecular basis driving the Eis1 Mab substrate specificity, we aimed at resolving the crystal structure of the protein. Extensive screening for crystallization conditions using either the apo-or ACO-bound form of Eis1 Mab generated numerous crystals. However, only crystals of the (ACO)-bound form of Eis1 Mab allowed the collection of exploitable datasets. We solved the crystal structure by molecular replacement using Eis2 Mab as a search model and the model was refined to a resolution of 2.9 Å (Table 1). Most residues could be modeled except for the first 23 N-terminus amino acids and part of helix 2 in all monomers.

The overall structure of Eis1 Mab is a multimer formed by six monomers. This hexameric architecture is similar to the organization of proteins belonging to the GNAT superfamily and also to Eis2 Mab and to its homologues in M. tuberculosis (Eis Mtb ) and M. smegmatis (Eis Msm ). It consists of the superposition of two homotrimers with a central vestibule (Figure 4A). This oligomeric state fully agrees with the apparent molecular mass determined by size-exclusion chromatography (Figure 2). The structure of the Eis1 Mab monomer can be divided into three subdomains: the Nterminal GNAT domain (residues 1-135), the central GNAT domain (residues 136-312) and the Cterminal domain (residues 313-415). Each monomer is composed of seventeen β-strands (β1 to β17), ten α-helices (α1 to α10) and seven 3 10 helices (η1 to η7) (Figure 4B).

4-The Acetyl-CoA binding site of Eis1 Mab

The best diffracting Eis1 Mab crystals were obtained in the presence of ACO. The simulatedannealed OMIT electron density map certifies the presence of the cofactor in the Eis1 Mab structure (Figure 4C). Due to the medium resolution, no water molecule could be modeled but fourteen residues involved in cofactor recognition could be identified (Figure 4D). While the side chains of Leu135 and Ile142 are involved in hydrophobic interactions, the His101 main chain provides a weak H-bond with the oxygen of the acetyl moiety. The carbonyl of Ile102 and Val104 establish also H-bonds. The ACO phosphate groups are recognized by the main chains of Arg110, Gly112, Ile114, and Thr115, as well as by the side chain of Thr115. Arg110 plays a key role in the interaction by making a salt-bridge with the phosphate group, creating a H-bond with the O group and a stacking interaction with the adenine ring of ACO. The carboxyl group of Glu139 binds to the NH2 of the adenine ring whereas Ser138 interacts with the O group of ACO. The Arg145 guanidium group recognizes the O4 of the ribose of ACO (Figure 4D). Additionally, multiple primary sequence alignments indicate that the cofactor binding site is relatively well conserved among the mycobacterial Eis proteins (Figure 5). Therefore, we rule out the possibility that these few amino acid substitutions in the cofactor binding site of Eis1 Mab would explain the difference of the substrate specificity between Eis1 Mab and the other Eis proteins.

5-The structural basis for Eis1 Mab substrate specificity

We next searched at the Protein Data Dank using the eFOLD (https://www.ebi.ac.uk/msdsrv/ssm/) and DALI 27 servers for the closest homologues of Eis1 Mab . First of all, we superposed Eis homologues structures from proteins that have been biochemically characterized and have been reported to modify aminoglycosides [START_REF] Green | Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria[END_REF][START_REF] Pricer | Biochemical and structural analysis of aminoglycoside acetyltransferase Eis from Anabaena variabilis[END_REF][START_REF] Green | Biochemical and structural analysis of an Eis family aminoglycoside acetyltransferase from bacillus anthracis[END_REF] . The multiple sequence alignments (Figure 5) and To understand the structural features responsible for Eis1 Mab 's preferences for small substrates, the crystal structure of Eis1 Mab was compared with those of Eis Mtb and Eis Msm which were solved in the presence of aminoglycosides. Eis Mtb was co-crystallized with tobramycin (PDB id: 4JD6) [START_REF] Houghton | Chemical and structural insights into the regioversatility of the aminoglycoside acetyltransferase Eis[END_REF] and Eis Msm with paromomycin (PDB id: 4QB9) [START_REF] Kim | Structure of Mycobacterium smegmatis Eis in complex with paromomycin[END_REF] . Most residues seen interacting with tobramycin in Eis1 Mab are not conserved except for the ultra-conserved carboxy-terminal Phe residue (Figure 5). Furthermore, among the eleven residues contacting paromomycin in Eis Msm , only three residues are semi-conserved (Thr100, His101, and Glu307) in Eis1 Mab while two others are strictly conserved (Ser138 and Phe415 at the C-terminus) (Figure 5). However, due to the partial conservation in the overall primary sequence between Eis Mtb , Eis Msm , and Eis2 Mab (Figure 5), it remains difficult to state that the inability of Eis1 Mab to modify aminoglycosides is inherent to the absence of amino acids conservation in the substrate-binding pocket. We, therefore, hypothesized that the size of Eis1 Mab active site pocket may be reduced as compared with other Eis proteins.

superposition
Determination of the central cavity volume that includes both the cofactor and substrate binding sites of each Eis protein was carried out with the CASTp server and, using its default settings [START_REF] Tian | CASTp 3.0: computed atlas of surface topography of proteins[END_REF] .

This analysis showed that Eis1 Mab has a rather small cavity of 651 Å which pushes further away helix 5. Consequently, this places the bulky side chains of residues Trp205, His210, and Phe213 closer to the Phe415 at the C-terminus, known to be required for substrate recognition (Figure 6C). These structural features reducing the volume of the Eis1 Mab substrate cavity are not seen in Eis Mtb , as the less bulky side chains of the equivalent residues Leu196, Leu200 and Glu202 are not protruding into the aminoglycoside-binding cavity (Figure 6C).

The structural comparison of the various Eis proteins unraveled an atypical feature in the central GNAT domain of Eis1 Mab (Figure 5 and Figure 6A). The region composed of fourteen residues and ranging from residues 235 to 248 (235-PQDTAEWFSSSART-248) is forming a loop and a 3 10 helix (η4, Figure 4B). This region spanning between strands β9 and β10 is extended in Eis1 Mab as compared with the other Eis proteins. This loop seems to act as a lid covering the substrate-binding site of the neighbor subunit (Figure 7A). Hence, this region might restrict the accessibility of the substrate-binding pocket to bulky molecules. Furthermore, Phe242, which is not conserved in other Eis homologues (Figure 5) seems of particular interest since its bulky side chain is protruding into the substrate-binding pocket, as can be appreciated when Eis1 Mab is superposed onto the tobramycin-bound Eis Mtb structure (Figure 7A). This may further reduce the volume of the substrate-binding site, thus compromising the access to large substrates, like aminoglycosides. Of note, this loop between strands β9 and β10 as well as Phe242 is strictly conserved in all subspecies of the M. abscessus complex (data not shown). Moreover, the corresponding loop was found to be much shorter in all the Eis proteins for which the enzymatic activity towards aminoglycosides was experimentally demonstrated. The loop of Eis Mtb or Eis Msm possesses 8 residues (228-VDRTDLKL-235) and 7 residues (226-RGPDGRR-232), respectively (Figure 7B). Additionally, even shorter loops are seen in two other Eis proteins whose activity towards aminoglycosides was demonstrated [START_REF] Green | Biochemical and structural analysis of an Eis family aminoglycoside acetyltransferase from bacillus anthracis[END_REF][START_REF] Pricer | Biochemical and structural analysis of aminoglycoside acetyltransferase Eis from Anabaena variabilis[END_REF] the loops in the Eis Ban (219-ENYK-222) and Eis Ava (221-RTRDGS-226) crystal structures are made of 4 and 6 residues, respectively (Figure 7B).

Discussion

Mycobacterial N-acetyltransferases and particularly Eis proteins play important roles in macrophage invasion, persistence, modulation of the immune response and antibiotic resistance [START_REF] Sanz-García | Mycobacterial Aminoglycoside Acetyltransferases: A Little of Drug Resistance, and a Lot of Other Roles[END_REF] .

Eis proteins modify a large set of aminoglycosides and have an impact on antibiotic resistance in numerous mycobacterial species, notably in M. tuberculosis and in NTM, including M. abscessus [START_REF] Ung | Crystal structure of the aminoglycosides N-acetyltransferase Eis2 from Mycobacterium abscessus[END_REF][START_REF] Rominski | Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes[END_REF] and M. fortuitum [START_REF] Sanz-García | Mycobacterial Aminoglycoside Acetyltransferases: A Little of Drug Resistance, and a Lot of Other Roles[END_REF] . Thus, molecules inhibiting Eis proteins given in combination with aminoglycosides would broaden the efficacy of this class of antibiotics in the fight against mycobacterial infections [START_REF] Ngo | Potent 1,2,4-Triazino[5,6 b]indole-3-thioether Inhibitors of the Kanamycin Resistance Enzyme Eis from Mycobacterium tuberculosis[END_REF] . The other biological functions of Eis protein related to the enhanced intracellular survival or to the modulation of the immune response of the host are also of interest in terms of druggability. Despite being closely related, the contribution of Eis1 Mab and Eis2 Mab to the intracellular survival of M. abscessus differs. While the deletion of eis1 Mab only slightly impairs the survival of M. abscessus in macrophages, the deletion of eis2 Mab profoundly affects the intracellular survival of the mutant strain [START_REF] Dubois | Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages[END_REF] . Additionally, regulation of the intracellular expression of the two genes is opposite as eis1 Mab is downregulated while eis2 Mab is upregulated in M. abscessus-infected macrophages [START_REF] Dubois | Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages[END_REF] . Importantly, our study unambiguously establishes that in contrast to Eis2 Mab , Eis1 Mab is not implicated in resistance to aminoglycosides, supporting previous findings [START_REF] Green | Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria[END_REF][START_REF] Rominski | Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes[END_REF][START_REF] Hurst-Hess | Mycobacterium abscessus WhiB7 Regulates a Species-Specific Repertoire of Genes To Confer Extreme Antibiotic Resistance[END_REF] . This absence of redundant function between the two Eis homologues makes Eis2 Mab as an interesting drug target to explore further to tackle drug resistance and virulence of M. abscessus.

Solving the crystal structure of Eis1 Mab sheds new light into the differential biological functions of Eis proteins in general and Eis1 Mab and Eis2 Mab more particularly. Structural comparison of many Eis structures belonging to the Mycobacterium or to other phyla identified key structural elements that are likely to block the access of Eis1 Mab binding site to large substrates such as aminoglycosides. In particular, the existence of an extended loop acting as a lid and covering the active site of the neighbor monomer as well as the positioning of helices 4 and 5 restrict the accessibility to the active site. Future work should be dedicated to investigating in more detail the substrate specificity of Eis1 Mab by creating defined mutants in the extended loop and/or α4-α5

helices. This appears achievable as modulation of the Eis Mtb 's substrate specificity was successfully done by mutating bulky residues that restrict the entrance of the active site [START_REF] Jennings | Redesign of substrate specificity and identification of the aminoglycoside binding residues of Eis from Mycobacterium tuberculosis[END_REF] .

This work may also add meaningful information allowing to predict the Eis-like protein substrate specificity. Like M. abscessus, other mycobacterial species possess several paralogues of the Eis protein [START_REF] Sanz-García | Mycobacterial Aminoglycoside Acetyltransferases: A Little of Drug Resistance, and a Lot of Other Roles[END_REF] , and predicting the function of these proteins particularly in the case of pathogenic bacteria may help to anticipate whether these Eis-like proteins may play a role in resistance to aminoglycosides. In that regard, bioinformatics tools such as multiple primary sequence alignments combine with protein structure homology modeling, could be useful to estimate the size of the loop between strands 9 and 10 (or strands 10 and 11 depending of the Eis origin) of the Eis homologues and consequently their substrate specificity.

While Eis1

Mab cannot modify aminoglycosides, our results indicate that it is an active Nacetyltransferase towards the small TYR and HIS arylalkylamine compounds, which are very unlikely the "natural substrates" of the enzyme. In the quest of identifying possible substrate of Eis1 Mab we carefully analyzed the genomic environment around eis1 Mab , and found that it probably belongs to an operon that is extremely well conserved among all the three M. abscessus subspecies and in other NTM, such as Mycobacterium chelonae, Mycobacterium saopaulense or Mycobacterium immunogenum. This operon also encompasses genes coding for a putative permease dibenzothiophene, an organosulfur compound found in petroleum [START_REF] Bhanjadeo | Differential desulfurization of dibenzothiophene by newly identified MTCC strains: Influence of Operon Array[END_REF] . Interestingly, the existence of such degradation pathway has also been described in Mycobacterium phlei [START_REF] Kayser | Isolation and characterization of a moderate thermophile, Mycobacterium phlei GTIS10, capable of dibenzothiophene desulfurization[END_REF] , which also possesses one Eis-like protein. Although Eis-like N-acetyltransferases have not been found yet functionally linked to such degradation pathway, this conserved genomic organization suggests that Eis1 Mab might be a new partner of this organosulfur chemicals degradation/detoxification pathway. This may be relevant to the fact that NTMs are environmental bacteria, which are likely to encounter organosulfur compounds in their ecological niche. In addition, that dibenzothiophene derivatives are also potent inhibitors of M. tuberculosis 39 may suggest that the eis1 Mab operon participates in resistance of M. abscessus to these compounds. Figure 2

A P R _ E i s 1 H Y G _ E i s 1 K A N _ E i s 1 Z E O _ E i s 1 A M K _ E i

  Inactivation of eis1 in M. abscessus.Deletion of MAB_4124 (eis1 Mab ) and MAB_4532c (eis2 Mab ) in the reference strain M. abscessus CIP104536 was carried out using the double homologous recombination unmarked strategy, reported previously[START_REF] Richard | Mutations in the MAB_2299c TetR Regulator Confer Cross-Resistance to Clofazimine and Bedaquiline in <em>Mycobacterium abscessus<[END_REF] . The DNA sequences located upstream and downstream the eis1 Mab gene were PCR-amplified and subsequently cloned into the pUX1-katG using the following primers and containing specific restriction sites (capital letters): fw_up_eis1_NheI 5'-The DNA sequences located upstream and downstream the eis2 gene were PCR-amplified and subsequently cloned into the pUX1-katG plasmid using the following primers and containing specific restriction sites (capital letters): fw_up_eis2_HpaI: 5'-catccGTTAACctggagctgctccgatgagccgaaatatgc-3'; rv_up_eis2_MfeI: 5'-The resulting plasmids were introduced by electroporation in the smooth (S) variant of M. abscessus and the red fluorescent, kanamycin (KAN)-resistant colonies which underwent the first homologous recombination event were selected on Middlebrook 7H10 agar supplemented with 10% Oleic acid-Albumin-Dextrose-Catalase (OADC) in the presence of 200 µg.mL -1 KAN. Cultures of selected colonies were then subjected to a counter-selection on 7H10 agar plates supplemented with OADC containing 50 µg.mL -1 isoniazid (INH) to promote a second homologous recombination and removal of the KAN cassette. Non-fluorescent, INH-resistant colonies were picked up and further genotyped for eis1 Mab deletion following PCR using two pairs of primers: fw_up_eis1_NheI with SQ2_b_down: 5'-cttactagaggagccatgggttctc-3' and SQ2_a_down: 5'-cgccaactccgttggtttggatatc-3'

5 '- 3 '

 53 -ctcaaCATATGatgTGGAGCCACCCGCAGTTCGAGAAGatggccttcaccgcgcagcctcaatggcc-3' and rv_Eis1_261_HindIII 5'-caatgAAGCTTtcagaagttggtacctgcgaaaggttgc-3' and subsequently cloned into pMV261-Zeo 21 in which the kanamycin cassette has been replaced by a zeocine resistance cassette, between NdeI and HindIII. We used similar strategy to complement the Δeis2 Mab mutant by cloning the eis2 gene into pMV261-Zeo using the following primers (restriction sites in capital and rv_Eis2_261_HindIII 5'-caatgAAGCTTtcagaagtcgtcgggcgcactgggtgc-3'.

  , ZEO (Zeocin), AMK (Amikacin), HYG (Hygromycin B), TYR (Tyramine), HIS (Histamine), acetyl-CoA (ACO) and enzymes were dissolved and diluted in buffer F. The premix A (enzyme, ACO, DTNB) and premix B (DTNB, substrate) were first incubated for 5 min at 25 o C.

  Crystals were grown in sitting drops in MR Crystallization Plates™ (Hampton Research) at 18 °C by mixing 2 µL of protein solution concentrated to 0.4 mg.mL -1 and containing 2 mM ACO with 2 µL of reservoir solution consisting of 0.8 M ammonium sulfate, 0.1 M Bis-Tris pH 5.5 and 1% (w/v) PEG 3350. Crystals were grown for one month and cryoprotected by a brief soaking step into a solution made of 1.4 M ammonium sulfate, 0.1 M Bis-Tris pH 5.5, 1% (w/v) PEG 3350 and 10% glycerol (v/v) prior being cryo-cooled in liquid nitrogen. X-ray diffraction data were collected at the Swiss-light source on the X06DA-PXIII beamline.

(

  MAB_4122), a predicted monooxygenase/acyl-CoA dehydrogenase (MAB_4123) located upstream of eis1 Mab and a nitrilotriacetate monooxygenase (MAB_4125) possessing a bacterial luciferase predicted domain, downstream of eis1 Mab . MAB_4122 and MAB_4125 are close homologues of the dibenzothiophene desulfurization enzymes A and C present in numerous bacteria able to degrade

Figure 1 :A

 1 Figure 1: Aminoglycosides susceptibility assessment of Eis1-overexpressing M. abscessus strains.

Figure 2 :

 2 Figure 2: Determination of the oligomeric state of Eis1 Mab Estimation of the oligomeric state of recombinant Eis1 Mab by size-exclusion chromatography (left panel). The elution profile of the proteins used for calibration is displayed as a black line and the elution profile of Eis1 Mab is shown in red. The calibration curve was established using β-amylase (1) (200 kDa), bovine serum albumin (2) (66 kDa), carbonic anhydrase (3) (29 kDa) and cytochrome C (4) (12.4 kDa), eluted with estimated volumes of 11.02, 13.2, 15.8 and 17.4 mL, respectively. The void volume corresponds to the dextran blue elution peak at 8.5 mL. The elution peak of Eis1 Mab at 10.9 mL matches an apparent molecular weight of 198 kDa. Coomassie Blue-stained SDS polyacrylamide gel electrophoresis of Eis1 Mab produced after the last step of purification on sizeexclusion chromatography (10 µg of protein; right panel).

Figure 3 :AC

 3 Figure 3: Biochemical characterization and enzymatic activity of Eis1 Mab. A Chemical structures of the different substrates tested in the assay. The structure of zeocin which is a very large molecule is not displayed. B Initial rates of Eis1 Mab determined in the presence of various substrates, including apramycin, hygromycin, kanamycin, zeocin, tyramine and histamine at a concentration of 5 mM. As a positive control, purified Eis2 Mab was also tested with TYR and HIS. C Michaelis and Menten curves used to determine the kinetic constants for TYR (left panel) and ACO (right panel).

Figure 4 :A

 4 Figure 4: Overall three-dimensional structure of Eis1 Mab . A Overall crystal structure of Eis1 Mab bound to ACO. The structure of the Eis1 Mab hexamer is depicted as a cartoon representation where each chain is colored differently. The left panel is a top view of the structure and the right one is rotated by 90°. ACO is pictured in each chain as a sphere representation. B Cross-eye stereo view of the Eis1 Mab monomer. The three domains are displayed as a cartoon representation where the N-terminal (Nt) GNAT is displayed as raspberry color, the central GNAT domain is in blue and the C-terminal (Ct) GNAT domain is in yellow. α, β and η indicate α-helices, β-strands, and 3 10 helices respectively. C Fo-Fc simulated-annealed OMIT map. The electron density map surrounding the ACO ligand is displayed as a grey mesh and contoured at 2.8 σ level. D ACO interactions with the Eis1 residues are shown as white sticks.Hydrogen-bond and the salt-bridge interactions are shown as black dashed lines.

Figure 5 :

 5 Figure 5: Structural comparison of mycobacterial Eis proteins. The multiple sequence alignment was performed with ENDscript 40 and adjusted manually. The secondary structure (α, α-helix; β, β-strand, η, 3 10 -helix) of Eis1 Mab extracted from its crystal structure, is indicated above the alignment. Residues involved in ACO binding in Eis1 Mab are indicated by the green circles, residues involved in aminoglycoside recognition in M. tuberculosis (Eis Mtb ) and M. smegmatis (Eis Msm ) Eis are depicted by the black and cyan square respectively.

Figure 6 :A

 6 Figure 6: Structural comparison of Eis proteins reveals unique features in Eis1 Mab . A Superposition of Eis crystal structures. All the structures are displayed as a ribbon representation with the following color codes: Eis1 Mab in slate, Eis Mtb in yellow, Eis2 Mab in raspberry, Eis Msm in magenta, Eis Ban in cyan, Eis Ava in orange, Eis Efa in salmon and Eis Kfl in red. Superposition of the carbon α was performed in Coot. The arrow indicates the loop between strands 9 and 10 which appears particularly extended in Eis1 Mab . B Comparison of the volume (grey surface area) of the active sites of the structures of Eis proteins that have been biochemically characterized, the color code is the same as in A. For structures solved with either ACO or CoA, the cofactor is shown as yellow sticks. C Key structural features restricting the size of the Eis1 Mab (slate) substrate-binding pocket. Arrows highlight the helices α4 and α5 movements seen in Eis1 Mab as compare with Eis Mtb (in yellow). Eis1 Mab residues side chains that protrude towards the substrate binding-site are shown

Figure 7 :

 7 Figure 7: Comparison of the extended loop size and conformation A Close up at the interface of two Eis1 Mab monomers. The extended loop of monomer 2 in slate is covering the active site of monomer 1 (in white) and the Phe242 side chain is pointing towards the substrate-binding site of monomer 1 that is close to the ACO (stick representation) binding pocket. The superposition of Eis Mtb (PDB id: 4JD6) bound to tobramycin in yellow, to Eis1 Mab highlights the steric hindrance of Phe242, presumably preventing the binding of aminoglycosides. B Comparison of the same loop region with the one in other Eis crystal structures from various bacterial origins. Eis Mtb is in yellow, Eis Msm in magenta, Eis Ban in cyan and, Eis Ava in orange.

  FIGURE 3

  acid sepharose column was used to adsorb the His-tagged TEV protease, the cleaved tag and the uncleaved tagged protein. The tag-free protein that weakly binds to the column was collected by

	buffer E (20 mM Tris-HCl pH 8, 0.2 M NaCl, 1 mM β-mercaptoethanol). A Nickel-Nitrilotriacetic
	An overnight culture of
	Escherichia coli BL21 (DE3) strain resistant to Phage T1 (New England Biolabs, Evry, France)
	carrying pRARE2 and transformed with pET41-eis1 Mab was grown under agitation at 180 rpm at 37
	o C in LB medium supplemented with 50 µg.mL -1 KAN and 30 µg.mL -1 chloramphenicol. This
	preculture was used to inoculate 6 L of LB broth supplemented with the same antibiotics at 37 o C.
	The cultures were cooled down in ice for 30 min when OD 600 reached ~0.8 and protein expression
	was induced with 1 mM of Isopropyl-β-D-thiogalactoside (IPTG) (Euromedex) for 16 h at 18 °C.
	Cell pellets were collected by centrifugation at 6, 000 g for 20 min and resuspended in buffer A (50
	mM Tris-HCl pH 8, 0.4 M NaCl, 1 mM β-mercaptoethanol, and 1 mM benzamidine). Bacteria were
	lysed by sonication and cell lysates were clarified by centrifugation at 28, 000 g at 4 o C for 1 h. The
	supernatant was then incubated 30 min with GST-sepharose beads with gentle agitation before
	being loaded onto a gravity column. Beads were washed with 10 column volumes of buffer B (50
	mM Tris-HCl pH 8, 0.4 M NaCl, 1 mM β-mercaptoethanol) and buffer C (50 mM Tris-HCl pH 8, 1
	M NaCl, 1 mM β-mercaptoethanol). Proteins were finally eluted with buffer D (50 mM Tris-HCl
	pH 8, 0.2 M NaCl, 1 mM β-mercaptoethanol and 15 mM glutathione). The eluate was incubated
	with TEV protease (1 mg of TEV protease per 40 mg of protein) and dialyzed overnight against

  of the Eis1 Mab monomer to the other Eis:ACO or Eis:CoA bound structures (Figure Anabaena variabilis, Eis Ava , (PDB id: 2OZG, 20% and r.m.s.d. 2.2 Å), unpublished. Additionally, the structure of Eis Ban from Bacillus anthracis (PDB id: 3N7Z) 29 solved without cofactor displays

	6A) generated the following primary sequence identity and root mean square deviation (r.m.s.d.)
	values: Eis Mtb (PDB id: 3RYO, 30% identity, r.m.s.d. 1.8 Å) 30 , Eis Msm (PDB id: 3SXN, 31%
	identity and r.s.m.d. 1.8 Å) 30 , Eis2 Mab (PDB id: 6RFT, 31% seq identity, r.m.s.d. 2.1 Å) 18 and

low sequence identity, 17% but a similar r.s.m.d of 2.1 Å. Our search allowed the comparison of two other Eis homologues that were not biochemically characterized, for instance, the Eis Efa from Enterococcus faecalis, with no ligand (PDB id: 2I00, 17%, r.m.s.d. 2.10 Å), and Eis Kfl from Kribbella flavida (PDB id: 4MY0, 21%, r.m.s.d. 2.4 Å). This structural comparison indicate that the closest homologues of Eis1 Mab are Eis proteins from the mycobacterium phylum.

  Mab with Eis Mtb , which possesses the largest cavity allows proposing that the small cavity in Eis1 Mab results of the positioning of helix 4

[START_REF] Duan | Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3[END_REF]

in comparison with the other Eis members that were experimentally shown to acetylate aminoglycosides (2005 Å 3 for Eis Mtb , 1678 Å 3 for Eis Msm , 1371 Å 3 for Eis Ava and 1274 Å 3 for Eis2 Mab ). Only Eis Ban has a slightly bigger volume than Eis1 Mab (695 Å 3 ) (Figure

6B

). Comparison of Eis1

Acknowledgements

We thank the staff at SLS beamlines for support during data collection, the Fondation pour la Recherche Médicale (FRM) [grant number DEQ20150331719 to LK], and A. Viljoen for fruitful discussion. KLU Ph.D. fellowship is supported by the National Research Agency [ANR-17-CE11-0008-01 -MyTraM to MB]

Conflict of interest statement

The authors declare no conflict of interest.

Author contributions