Jean Causse

Olivier Thomas

Aude-Valérie Jung

Marie-Florence Thomas
email: marie-florence.thomas@ehesp.fr

Direct DOC and nitrate determination in water using dual pathlength and second derivative UV spectrophotometry

Keywords: UV spectrophotometry, second derivative, nitrate, DOC, freshwaters, dual optical pathlength

come

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spectrophotometric measurement of raw samples (without filtration) coupling a dual pathlength for spectra acquisition and the second derivative exploitation of the signal is proposed in this work. The determination of nitrate concentration is carried out from the second derivative of the absorbance at 226nm corresponding at the inflexion point of nitrate signal decrease. A short optical pathlength can be used considering the strong absorption of nitrate ion around 210nm. For DOC concentration determination the second derivative absorbance at 295nm is proposed after nitrate correction. Organic matter absorbing slightly in the 270-330nm window, a long optical pathlength must be selected in order to increase the sensitivity. The method was tested on several hundred of samples from small rivers of two agricultural watersheds located in Brittany, France, taken during dry and wet periods. The comparison between the proposed method and the standardised procedures for nitrate and DOC measurement gave a good adjustment for both parameters for ranges of 2-100 mg/L NO3 and 1-30 mg/L DOC.

Introduction

Nutrient monitoring in water bodies is still a challenge. The knowledge of nutrient concentrations as nitrate and dissolved organic carbon (DOC) in freshwater bodies is important for the assessment of the quality impairment of water resources touched by eutrophication or harmful algal blooms for example. The export of these nutrients in freshwater is often characterized on one hand, by a high spatio-temporal variability regarding seasonal change, agricultural practices, hydrological regime, tourism and on the other hand, by the nature and mode of nutrient sources (punctual/diffuse, continuous/discontinuous) [START_REF] Causse | Variability of N export in water: a review[END_REF]. In this context the monitoring of nitrate and DOC must be rapid and

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easy to use on the field and UV spectrophotometry is certainly the best technique for that, given the great number of works, applications and systems proposed in the last decades.

Nitrate monitoring with UV sensing is a much more mature technique than DOC assessment by UV because nitrate ion has a specific and strong absorption. Several methods are available for drawing a relationship between UV absorbance and nitrate concentration using wavelength(s) around 200-220 nm, usually after sample filtration to eliminate interferences from suspended solids. Considering the presence of potential interferences such as dissolved organic matter (DOM) in real freshwater samples, the use of at least two wavelengths increases the quality of adjustment. The absorbance measurement at 205 and 300 nm was proposed by [START_REF] Edwards | Determination of Nitrate in Water Containing[END_REF] and the second derivative absorbance (SDA) calculated from three wavelengths was promoted by Suzuki and Kuroda (1987) and [START_REF] Crumpton | Nitrate and organic N analysis with 2nd-derivative spectroscopy[END_REF]. A comparison of the two methods (two wavelengths and SDA) carried out on almost 100 freshwater samples of different stations in a 35 km 2 watershed, gave comparable data with ion chromatography analysis (Olsen, 2008). Other methods based on the exploitation of the whole UV spectrum were also proposed in the last decades, namely for wastewater and sea water, with the aim of a better treatment of interferences. Several multiwavelength methods were thus designed such as the polynomial modelisation of UV responses of organic matter and colloids (Thomas et al.,1990), a semi deterministic approach, including reference spectra (nitrate) and experimental spectra of organic matter, suspended solids and colloids (Thomas et al., 1993), or partial least square regression (PLSR) method built-into a field portable UV sensor (Langergraber et al., 2003). Kröckel et al. (2011) for groundwater monitoring. The findings were that the MWS offers more possibilities for calibration and error detection, but requires more expertise compared with the DWS.

Contrary to UV measurement of nitrate in water, DOC is associated with a bulk of dissolved organic matter (DOM) with UV absorption properties less known and defined than nitrate.

The study of the relation between absorbing DOM (chromophoric DOM or CDOM) and DOC has given numerous works on the characterisation of CDOM by UV spectrophotometry or fluorescence on one hand, and on the assessment of DOC concentration from the measurement of UV parameters on the other hand. Historically the absorbance at 254nm (A254), 254 nm being the emission wavelength of the low pressure mercury lamp used in the first UV systems, was the first proxy for the estimation of Total organic carbon [START_REF] Dobbs | The use of ultra-violet absorbance for monitoring the total organic carbon content of water and wastewater[END_REF], and was standardised in 1995 [START_REF] Eaton | Measuring UV absorbing organics: a standard method[END_REF]. Then the specific UV absorbance, the ratio of the absorbance at 254 nm (A254) divided by the DOC value was also standardized ten years after (Potter and Wimsatt, 2005). Among the more recent works, Spencer et al. (2012) shown strong correlations between CDOM absorption (absorbance at 254 and 350 nm namely) and DOC for a lot of samples from 30 US watersheds. [START_REF] Carter | Freshwater DOM quantity and quality from a two-component model of UV absorbance[END_REF] proposed a two component model, one absorbing strongly and representing aromatic chromophores and the other absorbing weakly and associated with hydrophilic substances. After calibration at 270 and 350 nm, the validation of the model for DOC assessment was quite satisfactory for 1700 filtered surface water samples from North America and the UK. This method was also used for waters draining upland catchments and it was found that both a single wavelength proxy (263 nm or 230 nm) and a two wavelengths model performed well for both pore water and surface water (Peacock et al., 2014). Besides these one or two wavelengths methods, the use of chemometric ones was also proposed at the same time as nitrate determination from a same spectrum acquisition (Thomas et al., 1993); (Rieger et al., 2004) [START_REF] Avagyan | Application of high-resolution spectral absorbance measurements to determine dissolved organic carbon concentration in remote areas[END_REF] from the signal of a UV-vis submersible sensor with the recommendation to create site-specific calibration models to achieve the optimal accuracy of DOC quantification.

Among the above methods proposed for UV spectra exploitation, the second derivative of the absorbance (SDA) is rather few considered even if SDA is used in other application fields to enhance the signal and resolve the overlapping of peaks [START_REF] Bosch Ojeda | Recent applications in derivative ultraviolet/visible absorption spectrophotometry: 2009-2011[END_REF]. Applied to the exploitation of UV spectra of freshwaters SDA is able to suppress or reduce the signal linked to the physical diffuse absorbance of colloids and particles and slight shoulders can be revealed (Thomas and Burgess, 2007). If SDA was proposed for nitrate [START_REF] Crumpton | Nitrate and organic N analysis with 2nd-derivative spectroscopy[END_REF], its use for DOC has not been yet reported as well as a simultaneous SDA method for nitrate and DOC determination of raw sample (without filtration). This can be explained by the difficulty to obtain a specific response of organic matter and nitrate, in particular in the presence of high concentration of nitrate or high turbidity that cause spectra saturation and interferences. In this context, the aim of this work is to propose a new method to optimize the simultaneous measurement of DOC and nitrate using both dual optical pathlength and second derivative UV spectrophotometry.

Material and methods

Water samples

Water samples were taken from the Ic and Frémur watersheds (Brittany, France) through very different conditions during the hydrological year 2013-2014. These two rural watersheds of 86 km² and 77 km² respectively are concerned by water quality alteration with risks of green algae tides, closures of some beaches and contamination of seafood at their outlet. 580 samples were taken from spot-sampling (342 samples) on 32 different subwatersheds by dry or wet weather (defined for 5 mm of rain or more, 24 h before sampling) and by auto-

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sampling (233 samples) during flood events on 3 subwatersheds. For wet weather, sampling was planned by spot-sampling before the rain, or programmed according to the local weather forecast to ensure a sample collection proportional to the flow. Samples were collected in 1L polyethylene bottles (24 for auto-sampler ISCO 3700) following the best available practices.

Samples were transported to the laboratory in a cooler and stored at 5 ି ା 3°C (NF EN ISO 5667-3, 2013).

Data acquisition

Nitrate concentration was analyzed according to NF EN ISO 13395 standard thanks to a continuous flow analyzer (Futura Alliance Instrument). Dissolved organic carbon (DOC) was determined by thermal oxidation coupled with infrared detection (Multi N/C 2100, Analytik Jena) following acidification with HCl (standard NF EN 1484). Samples were filtered prior to the measurement with 0.45 µm HA Membrane Filters (Millipore®).

Turbidity (NF EN ISO 7027, 2000) was measured in situ for each sample, with a multiparameter probe (OTT Hydrolab MS5) for spot-sampling and with an Odeon probe (Neotek-Ponsel, France) for auto-sampling stations.

Finally discharge data at hydrological stations were retrieved from the database of the national program of discharge monitoring.

UV measurement

Spectra acquisition

UV spectra were acquired with a Perkin Elmer Lambda 35 UV/Vis spectrophotometer, between 200 and 400 nm with different Suprasil® quartz cells (acquisition step: 1 nm, scan speed: 1920 nm/min). Two types of quartz cells were used for each sample. A short path length cell of 2 mm was firstly used to avoid absorbance saturation in the wavelength domain strongly influenced by nitrate below 240 nm (linearity limited to 2.0 a.u.). On the contrary, a

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longer pathlength cell (20 mm) was used in order to increase the signal for wavelengths outside of the influence of nitrates (> 240 nm approx.). Regarding a classic UV spectrophotometer with a pathlength cell of 10 mm, these dual pathlength devices act as a spectrophotometric dilution/concentration system, adapted to a high range of variation of nitrate concentrations in particular.

Preliminary observation

Before explaining the proposed method, a qualitative relation between UV spectra shape and water quality can be reminded. Figure 1 shows two spectra of raw freshwaters with the same nitrate concentration (9.8 mgNO3/L) taken among samples of the present work. These spectra are quite typical of freshwaters. If the nitrate signal is well identified with the half Gaussian below 240 nm, the one of organic matter responsible for DOC is very weak with a very slight shoulder above 250 nm. In this context, the use of SDA already proposed for nitrate determination [START_REF] Crumpton | Nitrate and organic N analysis with 2nd-derivative spectroscopy[END_REF] and giving a maximum for any inflexion point in the decreasing part of the signal after a peak or a shoulder can be useful. However, given the absorbance values above 250 nm, the use of a longer optical pathlength is recommended in order to increase the sensitivity of the method.

Methodology

The general methodology is presented in Figure 2. Firstly, a UV spectrum is obtained directly from a raw sample (without filtration nor pretreatment) with a 2mm pathlength (PL) cell. If the absorbance value at 210 nm (A210), is greater than 2 u.a., a dilution with distilled water must be carried out. If not, the second derivative of the absorbance (SDA) at 226 nm is used for nitrate determination. The SDA value at a given wavelength λ is calculated according to the equation 1 (Thomas and Burgess 2007):

ܣܦܵ ఒ = ݇ * ൫ ഊష ା ഊశ -ଶ * ഊ ൯ మ [1]
where A λ is the absorbance value at wavelength λ, k is an arbitrary constant (chosen here equal to 1000) and h is the derivative step (here set at 10 nm).

Given the variability between successive SDA values linked to the electronic noise of the spectrophotometer, a smoothing step of the SDA spectrum is sometimes required, particularly when the initial absorbance values are low (< 0.1 a.u.). This smoothing step is based on the Stavitsky-Golay's method (Stavitsky and Golay, 1964).

For DOC measurement, the SDA value at 295 nm is used if A250 is greater than 0.1 a.u.. If A250 is lower than 0.1, the intensity of absorbances must be increased with the use of a 20mm pathlength cell. After the SDA295 calculation, a correction from the value of SDA226 linked to the interference of nitrate around 300nm is carried out. This point will be explained in the DOC calibration section. From the results of SDA values and the corresponding concentration of nitrate, a calibration is obtained for nitrate concentration ranging up to 100 mgNO 3 /L (Figure 4). This high value of nitrate concentration is possible thanks to the use of the 2 mm pathlength cell. Deduced from the calibration line, the R2 value is very close to 1 and the limit of detection (LOD) is 0.32 mgNO 3 /L. For DOC calibration, the procedure is different from the one for nitrate given the absence of standard solution for DOC, covering the complexity of dissolved organic matter. A test set of 49 samples was chosen among samples described hereafter, according to their DOC concentration up to 20 mgC/L. The choice of the SDA value at 295 nm is deduced from the examination of the second derivative spectra of some samples of the test set (Figure 5). Two peaks can be observed, the first one around 290 nm, and the second one less defined, around 330 nm (Figure 5a). The maximum of the first peak is linked to the DOC content, but its position shifts between 290 and 300 nm, because of the relation between DOC and nitrate concentration, with relatively more important SDA values when DOC is low (Thomas et al.

2014).

Considering that the measurement is carried out with a long optical pathlength for DOC, and that nitrate also absorbs in this region, its presence must be taken into account. On Figure 5a the second derivative spectrum of a 50 mgNO 3 /L of nitrate presents a valley (negative peak) around 310 nm and a small but large peak around 330 nm. Based on this observation, a correction is proposed for the SDA of the different samples (equation 2):

SDA* = SDAsample -SDAnitrate [2]
Where SDA* is the SDA corrected, SDA sample is the SDA calculated from the spectrum acquisition of a given sample and SDA nitrate is the SDA value corresponding to the nitrate concentration of the given sample.

After correction, the second derivative spectra show only a slight shift for the first peak around 300 nm and the peak around 330 nm is no more present (Figure 5b). From this observation, the SDA value at 295 nm is chosen for DOC assessment.

Samples characteristics

For this work, a great number of samples were necessary for covering the different subwatersheds characteristics and the variability of hydrometeorological conditions all along the hydrological year. 580 samples were taken from 32 stations and the majority of samples were taken in spring and summer time with regard to the principal land use of the two watersheds and the corresponding agricultural practices, namely fertilization (Figure 7).

Nitrate and DOC concentrations ranged respectively from 2.9 to 98.5 mgNO3/L and from 0.7 to 28.9 mgC/L. The river flows were between 0.8 L/s and 6299 L/s and turbidity between 0.1 and 821 NTU, after the rainy periods.

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Validation on freshwater samples

The validation of the method for nitrate determination was carried out on 580 samples (Figure 8). The adjustment between measured and estimated values of nitrate concentration gave a R 2 greater than 0.99 and a RMSE of 2.32 mgNO 3 /L. The slope is close to 1 and the ordinate is slightly negative (-1.68) which will be explained in the discussion section. The validation of the method for DOC determination was carried out on 580 samples (Figure 9). The adjustment between measured and estimated values of DOC concentration gave a R 2 greater than 0.95 and a RMSE close to 1 mgC/L. The slope is close to 1 and the ordinate is low (0.086 mgC/L).

Interferences

Except for DOC assessment for which the value of SDA at 295 nm must be corrected by the presence of nitrate, different interferences have to be considered in nitrate measurement.

Nitrate absorbing in the first exploitable window of UV spectrophotometry (measurement below 200 nm being quite impossible given the strong absorption of dioxygen) the presence of nitrite with a maximum of absorption at 213 nm could be a problem. However the molar absorption coefficient of nitrite is equal to the half of the value of nitrate (Thomas and Burgess, 2007) and usual concentrations of nitrite are much lower in freshwaters than nitrate ones (Raimonet et al., 2015). For other interferences linked to the presence of suspended solids or colloids (for raw samples) and organic matter for nitrate determination, Figure 10 shows some example of spectral responses for some samples of the tests set taken under contrasted conditions (dry and wet weather) and corrected from nitrate absorption (i.e. the contribution of nitrate absorption is deduced from the initial spectrum of a each sample). The spectral shape is mainly explained by the combination of physical (suspended solids and colloids) and chemical (DOM) responses. Suspended solids are responsible for a linear decrease of absorbance up to 350 nm and more, colloids for an exponential decrease between 200 and 240 nm and the main effect of the presence of DOM is the shoulder shape between 250 and 300 nm the intensity of which being linked to DOC content. Thus the spectral shape is not linear around the inflexion point of the nitrate spectrum (226 nm) and the corresponding second derivative values being low at 226 nm give a theoretical concentration under 2 mgNO 3 /L at maximum. This observation explains the slight negative ordinate of the validation curve (Figure 8). In order to confirm the need of nitrate correction of SDA at 295 nm for DOC determination the adjustment between DOC and SDA at 295nm without nitrate correction was carried out for the same set of samples than for DOC calibration (Figure 6). Compared to the characteristics of the corrected calibration line, the determination coefficient is lower (0.983 against 0.996) and the slope is greater (1.2 times) as well as the ordinate (7.9 times), the RMSE (5.6 times) and the LOD (5.4 against 1.1 mgNO3/L. These observations can be explained by the shift of the peak (around 290-295nm) and the hypochromic effect of nitrate on the SDA value of the sample at 295 nm, (see Figure 5) showing the importance of the nitrate correction for DOC determination.

Another interfering substance can be free residual chlorine absorbing almost equally at 200nm and 291nm with a molar absorption coefficient of 7.96*10 4 m 2 /mol at 291nm (Thomas O. and Burgess C., 2007), preventing the use of the method for chlorinated drinking waters.

Optical pathlength influence for NO3 and DOC

Two optical pathlengths are proposed for the method, a short one (2mm) for nitrate determination and a longer one (20mm) for DOC (Figure 2). However, considering that the optimal spectrophotometric range UV spectrophotometers between 0.1 and 2.0 a.u. (O Thomas and Burgess, 2007) must be respected, other optical pathlengths can be chosen for some water samples depending on their UV response. If the absorbance value of a sample is lower than 0.1 a.u. at 200nm with the 2mm optical pathlength, a 20mm quartz cell must be used. Respectively, if the absorbance value is lower than 0.1 at 300nm with the proposed optical pathlength of 20mm, a 100 mm one must be used. This can be the case when nitrate or DOC concentration is very low given the inverse relationship often existing between these two parameters (Thomas et al., 2014). A comparison of the use of different optical pathlength for DOC estimation gives an R 2 value of 0.70 for the short pathlength (2mm) against 0.96 for the recommended one (20mm). Finally, the choice of a dual pathlength measurement was recently proposed by [START_REF] Chen | Development of variable pathlength UV-vis spectroscopy combined with partial-least-squares regression for wastewater chemical oxygen demand (COD) monitoring[END_REF] to improve successfully the chemical oxygen demand estimation in wastewater samples by using a PLS regression model applied to the two spectra.

Conclusion

A simple and rapid method for the UV determination of DOC and nitrate in raw freshwater samples, without filtration, is proposed in this work:

-Starting from the acquisition UV absorption spectra with 2 optical pathlengths (2 and 20 mm), the second derivative values at 226 and 295 nm are respectively used for nitrate and DOC measurement.

-After a calibration step with standard solutions for nitrate and known DOC content samples for DOC, LODs of 0.3 mgNO3/L for nitrate and 1.1 mgC/L were obtained for ranges up to 100 mgNO3/L and 0-25 mgC/L. -Given its simplicity, this method can be handled without chemometric expertise and adapted on site with field portable UV sensors or spectrophotometers.

It is the first UV procedure based on the use of the second derivative absorbance at 295nm for DOC determination, and calculated after correction of nitrate interference from the acquisition of the UV absorption spectrum with a long optical pathlength (20mm or more). This is a simple way to enhance the slight absorption shoulder around 280-300nm due to the presence of organic matter. Moreover the interferences of suspended matter and colloids being negligible on the second derivative signal, the measurement can be carried out for both parameters on raw freshwater samples without filtration. Finally, even if the validation of the method was carried out on a high number of freshwater samples covering different hydrological conditions, further experimentations should be envisaged in order to check the applicability of the method to the variability of DOM nature.

  proposed a combined method of exploitation (multi component analysis (MCA) integrating reference spectra and a polynomial modelisation of humic acids), associated to a miniaturized spectrophotometer with a capillary flow cell. More recently, a comparison between two different commercials in situ spectrophotometers, a double wavelength spectrophotometer (DWS) and a M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT multiwavelength one (MWS) with PLSR resolution was carried out by Huebsch et al. (2015)

Figure 1 :

 1 Figure 1: Example of UV spectra of raw freshwaters with the same concentration of nitrate

  Figure 2 : General methodology

Figure 3

 3 Figure 3 shows spectra and second derivatives of standard nitrate solutions. Nitrate strongly absorbs around 200-210 nm with a molar absorption coefficient of 8.63*10 5 m 2 /mol at 205.6

Figure 3 :

 3 Figure 3 : Spectra of standard solutions of nitrate (raw absorbances left, and SDA right)

Figure 4 :

 4 Figure 4 : Calibration line for nitrate determination from SDA at 226 nm (R 2 being equal to 1,

  Figure5: Second derivative spectra of water samples without nitrate correction (5a with a

Fig 6 :

 6 Fig 6: Calibration between SDA at 295nm corrected by nitrate (SDA* 295 ) and DOC

Figure 7 :

 7 Figure 7: Relevance of samples in relation to land use, seasonality, land use and physic-

Fig 8 :

 8 Fig 8: Relation between measured and estimated (from SDA 226 ) NO 3 concentrations for 580

Fig 9 :

 9 Fig 9: Relation between measured and estimated (by SDA* 295 ) DOC concentrations for 580

Figure 10 :

 10 Figure 10: Spectra of freshwater samples corrected from nitrate absorbance. Nitrate, DOC and

  The peak around 295nm for the second derivative spectra reveals the existence of an inflexion point at the right part of the slight shoulder of the absorbance spectrum, between 250 and 300 nm. This observation can be connected with the use of the spectral slope between 265 and 305 nm(Galgani et al., 2011) to study the impact of photodegradation and mixing processes on the optical properties of dissolved organic matter (DOM) in the complement of fluorescence in two Argentine lakes. Fichot and Benner (2012) also used the spectral slope between 275 and 295 nm for CDOM characterisation and its use as tracers of the percent terrigenous DOC in river-influenced ocean margins.Helms et al. (2008) propose to consider two distinct spectral slope regions (275-295 nm and 350-400 nm) within log-transformed absorption spectra in order to compare DOM from contrasting water types. The use of the logtransformed spectra was recently proposed byRoccaro et al. (2015) for raw and treated drinking water and the spectral slopes between 280 and 350nm were shown to be correlated to the reactivity of DOM and the formation of potential disinfection by-products. Finally a very recent study(Hansen et al., 2016) based on the use of DOM optical properties for the discrimination of DOM sources and processing (biodegradation, photodegradation), focused on the complexity of DOM nature made-up of a mixture of sources with variable degrees of microbial and photolytic processing and on the need for further studies on optical properties of DOM. Thus, despite the high number of samples considered for this work and the contrasted hydrological conditions covered, the relevance of DOM nature as representing all types of DOM existing in freshwaters is not ensured. The transposition of the method, at least for DOC assessment, supposes to verify the existence of the second derivative peak at 290-300 nm and the quality of the relation between the SDA value at 295 (after nitrate correction), and the DOC content.

-

  The method validation was carried out for around 580 freshwater samples representing different hydrological conditions in two agricultural watersheds.

Acknowledgement

The authors wish to thank the Association Nationale de la Recherche et de la Technologie (ANRT), and Coop de France Ouest for their funding during the PhD of Jean Causse (PhD grant and data collection), the Agence de l'Eau Loire-Bretagne and the Conseil Régional de Bretagne for their financial support (project C&N transfert).

Etheridge, J.R., Birgand, F., Osborne, J. a., Osburn, C.L., Burchell Ii, M.R., Irving, J., 2014. Using in situ ultraviolet-visual spectroscopy to measure nitrogen, carbon, phosphorus, and suspended solids concentrations at a high frequency in a brackish tidal marsh. Limnol. Oceanogr. Methods 12, 10-22.

Fichot, C.G., Benner, R., 2012. The spectral slope coefficient of chromophoric dissolved organic matter (S275-295) as a tracer of terrigenous dissolved organic carbon in river-influenced ocean margins. Limnol. Oceanogr. 57, 1453-1466. Galgani, L., Tognazzi, A., Rossi, C., Ricci, M., Angel Galvez, J., Dattilo, A.M., Cozar, A., Bracchini, L., Loiselle, S.A., 2011. Assessing the optical changes in dissolved organic


M G Ferrarini

S G Mucha

D Parrot

G Meiffren

J F R Bachega

G Comt

A Zaha
email: zaha@cbiot.ufrgs.br

M F Sagot

Hydrogen peroxide production and myo-inositol metabolism as important traits for virulence of Mycoplasma hyopneumoniae

Keywords:

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia. In our previous work, we reconstructed the metabolic models of this species along with two other mycoplasmas from the respiratory tract of swine: Mycoplasma hyorhinis, considered less pathogenic but which nonetheless causes disease and Mycoplasma flocculare, a commensal bacterium. We identified metabolic differences that partially explained their different levels of pathogenicity. One important trait was the production of hydrogen peroxide from the glycerol metabolism only in the pathogenic species. Another important feature was a pathway for the metabolism of myo-inositol in M. hyopneumoniae. Here, we tested these traits to understand their relation to the different levels of pathogenicity, comparing not only the species but also pathogenic and attenuated strains of M. hyopneumoniae. Regarding the myo-inositol metabolism, we show that only M. hyopneumoniae assimilated this carbohydrate and remained viable when myo-inositol was the primary energy source. Strikingly, only the two pathogenic strains of M. hyopneumoniae produced hydrogen peroxide in complex medium. We also show that this production was dependent on the presence of glycerol. Although further functional tests are needed, we present in this work two interesting metabolic traits of M. hyopneumoniae that might be directly related to its enhanced virulence.

Contents

Introduction

The notion that the lungs are sterile is frequently stated in textbooks; however, no modern studies have provided evidence for the absence of microorganisms in this environment [START_REF] Dickson | The lung microbiome: New principles for respiratory bacteriology in health and disease[END_REF]. Several bacteria colonize the respiratory tract of swine.

Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis are some of the most important species identified so far [START_REF] Mare | New species: Mycoplasma hyopneumoniae; a causative agent of virus pig pneumonia[END_REF][START_REF] Meyling | Serological identification of a new porcine Mycoplasma species, Mycoplasma flocculare[END_REF][START_REF] Rose | Taxonomy of some swine Mycoplasmas: Mycoplasma suipneumoniae goodwin et al. 1965, a later, objective synonym of Mycoplasma hyopneumoniae mare and switzer 1965, and the status of Mycoplasma flocculare meyling and friis 1972[END_REF][START_REF] Siqueira | Microbiome overview in swine lungs[END_REF]. M. hyopneumoniae is widespread in pig populations and is the causative agent of enzootic pneumonia [START_REF] Maes | Enzootic pneumonia in pigs[END_REF]; M. hyorhinis, although not as pathogenic as M. hyopneumoniae, has already been found as the sole causative agent of pneumonia, polyserositis and arthritis in pigs [START_REF] Kobisch | Swine mycoplasmoses[END_REF][START_REF] Davenport | Polyserositis in pigs caused by infection with Mycoplasma[END_REF][START_REF] Whittlestone | Porcine mycoplasmas[END_REF][START_REF] Thacker | Mycoplasmosis[END_REF]. M. flocculare, on the other hand, has high prevalence in swine herds worldwide, but up to now, is still considered a commensal bacterium [START_REF] Kobisch | Swine mycoplasmoses[END_REF].

Because of the genomic resemblance of these three Mycoplasma species [START_REF] Stemke | Phylogenetic relationships of three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis, and complete 16S rRNA sequence of M. flocculare[END_REF][START_REF] Siqueira | New insights on the biology of swine respiratory tract mycoplasmas from a comparative genome analysis[END_REF], it remains unclear why M. hyopneumoniae can become highly virulent if compared with the other two. It is also essential to understand that the simple presence or absence of each species is not in itself a determinant factor in the development of enzootic pneumonia: most piglets are thought to be vertically infected with M. hyopneumoniae at birth [START_REF] Maes | Enzootic pneumonia in pigs[END_REF][START_REF] Fano | Assessment of the effect of sow parity on the prevalence of Mycoplasma hyopneumoniae in piglets at weaning[END_REF][START_REF] Sibila | Current perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection[END_REF] and many can become carriers of the pathogen throughout their entire life without developing acute pneumonia. Moreover, M. hyopneumoniae also persists longer in the respiratory tract, either in healthy animals or even after successful treatment of the disease [START_REF] Thacker | Interaction between Mycoplasma hyopneumoniae and swine influenza virus[END_REF][START_REF] Ruiz | Mycoplasma hyopneumoniae colonization of pigs sired by different boars[END_REF][START_REF] Fano | Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs[END_REF][START_REF] Overesch | Persistence of Mycoplasma hyopneumoniae sequence types in spite of a control program for enzootic pneumonia in pigs[END_REF].

To make it even more complex, different strains of each species bear different levels (or even lack) of pathogenicity. For instance, M. hyopneumoniae has six sequenced strains, two of which are known to be attenuated by culture passages [START_REF] Zielinski | Effect of growth in cell cultures and strain on virulence of Mycoplasma hyopneumoniae for swine[END_REF][START_REF] Liu | Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic 168 strain and its high-passaged attenuated strain[END_REF]. These strains cannot cause the clinical symptoms of pneumonia in vivo and up to now it is not clear why.

In contrast to other pathogenic bacteria, and as revealed by the analysis of the sequenced genomes from several mycoplasmas [START_REF] Himmelreich | Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae[END_REF][START_REF] Chambaud | The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis[END_REF][START_REF] Minion | The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis[END_REF][START_REF] Vasconcelos | Swine and poultry pathogens: the complete genome sequences of two[END_REF][START_REF] Siqueira | New insights on the biology of swine respiratory tract mycoplasmas from a comparative genome analysis[END_REF], pathogenic Mycoplasma species seem to lack typical primary virulence factors such as toxins, invasins, and cytolysins [START_REF] Pilo | A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony[END_REF][START_REF] Maes | Update on Mycoplasma hyopneumoniae infections in pigs: Knowledge gaps for improved disease control[END_REF]. For this reason, classical concepts of virulence genes are usually problematic and a broader concept for virulence is used for these species. In this way, a virulence gene in mycoplasmas is described as any non essential gene for in vitro conventional growth, which is essential for the optimal survival (colonization, persistence or pathology) inside the host [START_REF] Browning | Identification and characterization of virulence genes in mycoplasmas[END_REF].

There have been many different types of virulence factors described so far in several Mycoplasma species, most of them related to adhesion [START_REF] Razin | Mycoplasma adhesion[END_REF], invasion [START_REF] Burki | Virulence, persistence and dissemination of Mycoplasma bovis[END_REF], cytotoxicity [START_REF] Vilei | Genetic and biochemical characterization of glycerol uptake in Mycoplasma mycoides subsp. mycoides SC: its impact on H(2)O(2) production and virulence[END_REF][START_REF] Hames | Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae[END_REF], host-evasion [START_REF] Simmons | How Some Mycoplasmas evade host immune responses[END_REF] and host-immunomodulation [START_REF] Katz | Comparison of mitogens from Mycoplasma pulmonis and Mycoplasma neurolyticum[END_REF][START_REF] Waites | Mycoplasma pneumoniae and its role as a human pathogen[END_REF].

As for M. hyopneumoniae and M. hyorhinis, adhesion factors such as antigen surface proteins and the ability of these organisms to produce a capsular polysaccharide have already been described in the literature [START_REF] Whittlestone | Porcine mycoplasmas[END_REF][START_REF] Tajima | Interaction of Mycoplasma hyopneumoniae with the porcine respiratory epithelium as observed by electron microscopy[END_REF][START_REF] Citti | Elongated versions of Vlp surface lipoproteins protect Mycoplasma hyorhinis escape variants from growth-inhibiting host antibodies[END_REF][START_REF] Djordjevic | Proteolytic processing of the Mycoplasma hyopneumoniae cilium adhesin[END_REF][START_REF] Seymour | Mhp182 (P102) binds fibronectin and contributes to the recruitment of plasmin(ogen) to the Mycoplasma hyopneumoniae cell surface[END_REF]. However, while the diseases caused by these swine mycoplasmas have been extensively studied, only recently their metabolism has been explored from a mathematical and computational point of view by our group [START_REF] Ferrarini | Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling[END_REF]. We are well aware that metabolism does not fully explain the pathologies caused by either of them. However, adhesion proteins, classically related to virulence in mycoplasmas cannot be associated with the different levels of pathogenicity between M. hyopneumoniae and M. flocculare. Both species harbor similar sets of adhesion proteins [START_REF] Siqueira | Unravelling the transcriptome profile of the swine respiratory tract mycoplasmas[END_REF] and have been shown to adhere to cilia in a similar way [START_REF] Young | A tissue culture system to study respiratory ciliary epithelial adherence of selected swine mycoplasmas[END_REF]. Thus, it remains unclear what prevents M. flocculare to cause disease in this context.

In our previous work [START_REF] Ferrarini | Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling[END_REF], we compared the reconstructed metabolic models of these three Mycoplasma species, and pointed out important metabolic differences that could partly explain the different levels of pathogenicity between the three species. The most important trait was related to the glycerol metabolism, more specifically the turnover of glycerol-3-phosphate into dihydroxyacetone-phosphate (DHAP) by the action of glycerol-3-phosphate oxidase (GlpO, EC 1.1.3.21), which was only present in the genomes of M. hyorhinis and M. hyopneumoniae. This would allow the usage of glycerol as a primary energy source, with the production of highly toxic hydrogen peroxide in the presence of molecular oxygen. The metabolism of glycerol and the subsequent production of hydrogen peroxide by the action of GlpO are essential for the cytotoxicity of lung pathogens Mycoplasma pneumoniae [START_REF] Hames | Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae[END_REF] and Mycoplasma mycoides subsp. mycoides [START_REF] Vilei | Genetic and biochemical characterization of glycerol uptake in Mycoplasma mycoides subsp. mycoides SC: its impact on H(2)O(2) production and virulence[END_REF]. Moreover, the Mycoplasma hominis group is not the only one where hydrogen peroxide production via glpO has been reported. In some Spiroplasma species (specifically Spiroplasma taiwanense) and within the pneumoniae group (for instance in Mycoplasma penetrans), the presence of this enzyme was also associated with virulence [START_REF] Kannan | Hemolytic and hemoxidative activities in Mycoplasma penetrans[END_REF][START_REF] Lo | Comparison of metabolic capacities and inference of gene content evolution in mosquitoassociated Spiroplasma diminutum and S. taiwanense[END_REF].

Another major difference between our previous models was related to the presence of a complete transcriptional unit (TU) encoding proteins for the uptake and metabolism of myo-inositol in M. hyopneumoniae (with the exception of one enzyme). This could be another important trait for the enhanced virulence of this species if compared with the other two. Here, we studied this pathway in more detail to try to find this missing enzyme and the possible reasons as to why natural selection kept these genes only in this Mycoplasma species.

In a recent review, Maes and colaborators [START_REF] Maes | Update on Mycoplasma hyopneumoniae infections in pigs: Knowledge gaps for improved disease control[END_REF] emphasize the need for the further investigation of the role of glycerol and myo-inositol metabolism and their contribution to virulence in M. hyopneumoniae. Here, we experimentally tested these two traits to show how they might be related to the different levels of pathogenicity, by comparing not only the species themselves but different strains of M. hyopneumoniae. Contrary to what we anticipated, only the two pathogenic strains of M. hyopneumoniae were able to produce hydrogen peroxide in complex medium, and we confirmed that this production was dependent on the presence of glycerol. The myo-inositol metabolism, in turn, was tested with the aid of deuterated myo-inositol in Friis medium. We were able to detect by mass spectrometry (MS) a slight decrease in the marked myo-inositol concentration throughout time, indicating the ability of M. hyopneumoniae to uptake such carbohydrate. We also show here that only the M. hyopneumoniae strains remained viable when myo-inositol was the primary energy source.

We present here two metabolic traits specific to M. hyopneumoniae that might be directly related to its enhanced virulence, specially in its ability to successfully overgrow the other two Mycoplasma species in the respiratory tract of swine, persist longer in this environment and possibly cause disease.

Results

Comparative genomics of glpO from glycerol metabolism

Highly conserved homolog genes to glpO from M. mycoides subsp. mycoides (EC 1.1.3.21) were found only in the genomes of M. hyopneumoniae and M. hyorhinis. Despite the annotation as a dehydrogenase in both M. hyopneumoniae and M. hyorhinis, we propose this enzyme to act as glycerol-3-phosphate oxidase (GlpO), using molecular oxygen as the final electron acceptor and producing DHAP and hydrogen peroxide. We therefore refer to the encoded protein in M. hyopneumoniae and M. hyorhinis as GlpO, rather than GlpD. The high similarity between these predicted proteins (Supplementary Figure S1A) may be an indication that this trait might be essential for the pathogenicity of these Mycoplasma species.

Particularly, the cytotoxicity of M. mycoides subsp. mycoides is considered to be related to the translocation of the hydrogen peroxide into the host cells [START_REF] Bischof | Cytotoxicity of Mycoplasma mycoides subsp. mycoides small colony type to bovine epithelial cells[END_REF]. This is presumably possible because of the close proximity to the host cells along with the integral membrane GlpO [START_REF] Pilo | A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony[END_REF][START_REF] Pilo | Molecular mechanisms of pathogenicity of Mycoplasma mycoides subsp. mycoides SC[END_REF]. Different transmembrane prediction softwares [START_REF] Hofmann | TMbase -A database of membrane spanning proteins segments[END_REF][START_REF] Krogh | Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes[END_REF][START_REF] Combet | NPS@: network protein sequence analysis[END_REF][START_REF] Kahsay | An improved hidden Markov model for transmembrane protein detection and topology prediction and its applications to complete genomes[END_REF] identified putative transmembrane portions in the GlpO proteins from M. hyopneumoniae and M. hyorhinis (Supplementary Figure S1B). Similar results were reported for the homolog enzyme in M. mycoides subsp. mycoides [START_REF] Pilo | A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony[END_REF], and a recent proteomic study has detected GlpO from M. hyopneumoniae in surface-enriched extracts through LC-MS/MS (Personal communication from H. B. Ferreira, [START_REF] Machado | Comparative surface proteomic approach reveals qualitative and quantitative differences of two Mycoplasma hyopneumoniae strains and Mycoplasma flocculare[END_REF]).

Pathogenic M. hyopneumoniae strains produce hydrogen peroxide from glycerol

Contrary to what we had anticipated, we were only able to detect the production of hydrogen peroxide from the two pathogenic strains of M. hyopneumoniae (7448 and 7422) in Friis medium, as can be seen in Figure 1A. The attenuated strain from the same species (M. hyopneumoniae strain J), along with M. hyorhinis and M. flocculare did not produce detectable quantities of this toxic product. In order to verify if the amount of hydrogen peroxide produced was comparable between strains, we also counted the number of cells for each replicate. In this way, the two pathogenic strains produced approximately the same amount of hydrogen peroxide and had cell counts of the same order of magnitude (available in Supplementary Table S1).

We also show (Figure 1B) that the hydrogen peroxide produced by the M. hyopneumoniae strains 7448 and 7422 was dependent on the presence of glycerol in the incubation buffer.

Levels of glpO transcripts do not differ from pathogenic to attenuated strains of M. hyopneumoniae

We tested the three M. hyopneumoniae strains (7448, 7422 and J) in order to compare the mRNA expression levels of glpO gene by RT-qPCR. Since the transcript levels of normalizer genes were not comparable between strains, we used relative quantification normalized against unit mass; in our case, the initial amount of RNA. We chose one of the replicates from strain 7448 as the calibrator, and we were able to show (Figure 2 and Supplementary Table S2) that there was no significant difference in the transcript levels of glpO in all tested strains from M. hyopneumoniae.

Enzymes for the uptake and catabolism of myo-inositol are specific to M. hyopneumoniae strains

M. hyopneumoniae is the only reported species among the Mollicutes that contains genes involved in the catabolism of myo-inositol. Since Mycoplasma species seem to maintain a minimum set of essential metabolic capabilities, we decided to further investigate this pathway and the influence of its presence on the metabolism and pathogenicity of M. hyopneumoniae. The degradation of inositol can feed glycolysis with DHAP and also produces an acetyl coenzyme-A (AcCoA) (Figure 3). A TU for the myo-inositol catabolism is present in all M. hyopneumoniae strains, with the exception of the gene that codes for the enzyme 6-phospho-5-dehydro-2-deoxy-D-gluconate aldolase (IolJ, EC 4.1.2.29), responsible for the turnover of 6-phospho-5-dehydro-2-deoxy-D-gluconate (DKGP) into malonate semialdehyde (MSA).

The gene encoding IolJ in other organisms is similar to the one coding for enzyme fructose-bisphosphate aldolase (Fba) from glycolysis (EC 4.1.2.13).

There are two annotated copies of the gene fba in M. hyopneumoniae (fba and fba-1, Supplementary Table S3). We performed homology and gene context analyses, 3D comparative modeling and protein-ligand interaction analysis to check if either of them would be a suitable candidate for this activity.

The gene context and protein sequence alignment for 15 selected Fba homologs in Mollicutes can be seen in Supplementary Figures S2 andS3. Comparative models for both copies of Fba from M. hyopneumoniae and previously characterized IolJ and Fba from Bacillus subtilis [START_REF] Yoshida | myo-Inositol catabolism in Bacillus subtilis[END_REF] were constructed based on available structures of Fba in PDB [START_REF] Berman | The Protein Data Bank[END_REF] (Figure 4 and Supplementary Table S4).

Fba structures from Escherichia coli and Giardia intestinalis were used to gather more information about substrate binding (Supplementary Figure S3). The alignment shows a highly conserved zinc binding site (residues marked as '*'), essential for substrate binding and catalysis. Positions 'a', 'b', 'c', 'd' and 'e' surround the substrate cavity. The structural analysis suggests that the interaction mode of DKGP (substrate of IolJ) with the zinc ion of the active site is similar to that observed for FBP (fructose-1,6-bisphosphate, substrate of Fba).

Nevertheless the substrate specificity is strongly dependent on the residues that form the substrate cavity.

While there seems to be several common features between Fba and IolJ (residues 'c', 'd', 'e' and '*'), residue 'a' appears to be essential for the substrate interaction with IolJ. This residue is generally occupied by an arginine (R52) in several putative IolJs from other organisms (Supplementary Figure S4), and absent in all predicted Fbas analysed in this study. From the predicted structures, the presence of this positively charged arginine in IolJ seems to disfavour the interaction with the phosphate group of FBP whilst it is complementary to the carboxyl group from DKGP.

In this way, the predicted structure of Fba-1 from M. hyopneumoniae resembles more the Fba structures from the experimentally solved Fbas in B. subtilis, E. coli and G. intestinalis. The annotated Fba from M. hyopneumoniae, on the other hand, seems to be more similar to the IolJ structure from B. subtilis. Although functional studies are needed to test this hypothesis, we propose that all enzymes needed for the myo-inositol catabolism are present in M. hyopneumoniae.

M. hyopneumoniae is able to uptake myo-inositol from the culture medium

In order to ascertain the ability of different bacteria to uptake myo-inositol, we used two different approaches. The first was the use of marked myo-inositol in complex medium and analysis by MS, and the second was to check the viability of cells (through ATP production) whenever myo-inositol was used as primary energy source.

When we tested if cells were able to uptake the marked myo-inositol, over the course of 48 h, we found no significant difference in M. flocculare and M. hyorhinis when compared to the control medium (CTRL), as observed in Figure 5A. As expected, the concentrations of myo-inositol for both strains of M. hyopneumoniae after 48 h of growth were lower than the control medium. We also collected two extra time points for M. hyopneumoniae strain 7448 and CTRL: 8 h and 24 h of growth (Figure 5B). In all time points, there is significant difference between the residual marked myo-inositol and the control medium, which implies that M. hyopneumoniae is able to uptake such carbohydrate from the medium. MS peak data is available in Supplementary Table S5.

Since we had glucose and glycerol present in this complex medium analysed by MS, we also wanted to check if the viability of the different strains and species altered when myo-inositol was the primary energy source. For this, we incubated cells in myo-inositol defined medium (depleted with glucose and glycerol) for 8 hours and measured the amount of ATP these cells were able to produce. This in turn was directly related to the amount of viable cells after cultivation in the specific medium tested. Considering we do not know the energetic yield and efficiency of each strain and species, we could not directly compare the amount of ATP produced between different organisms. For this reason, growth in regular defined medium (with glucose) for each strain was used as a normalization control and the ratio of ATP production in both media was used to compare the viable cells between strains. Since there was no other energy source available in the medium and in accordance with our previous predictions and results, only M. hyopneumoniae cells remained viable (ranging from 75% to 280%) when compared to their control growth in the regular defined medium (Figure 6 and Supplementary Table S6). The viability of the other species in this medium was 11.5% for M. hyorhinis and 0.2% for M. flocculare. We also achieved similar results when comparing the growth in myo-inositol defined medium versus Friis medium (Supplementary Figure S5).

Discussion

In this study, we wanted to find possible differences between pathogenic and attenuated strains of M. hyopneumoniae and also compare them with M. hyorhinis and M. flocculare and assess possible links to the enhanced virulence of M. hyopneumoniae. While M. hyopneumoniae strains 7422 and 7448 are considered pathogenic, strain J became attenuated after serial passages of in vitro culture; M. hyorhinis strain ATCC 17981 was isolated from swine but, to our knowledge, its level of pathogenicity has not been tested in vivo; and even though M. flocculare is not considered pathogenic, strain ATCC 27399 was isolated from a case of swine pneumonia (strain ATCC 27716 is derived from this strain). In our previous study [START_REF] Ferrarini | Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling[END_REF], through mathematical modeling, we predicted two traits of M. hyopneumoniae in silico that could be associated with its enhanced virulence: the myo-inositol catabolism and the link between the glycerol and the glycolysis metabolism, with the production of highly toxic hydrogen peroxide (by the activity of the GlpO enzyme). In this work, we tested whether these species indeed differed from each other regarding their ability (i) to produce hydrogen peroxide in vitro and whether this was related to the availability of glycerol, (ii) to uptake myo-inositol, and (iii) to remain viable in a defined medium with myo-inositol as the primary energy source. While the uptake of myo-inositol might be a general feature of M. hyopneumoniae, the production of hydrogen peroxide in complex medium seems to be specific to pathogenic strains of this species.

Glycerol metabolism and hydrogen peroxide production

Even though the GlpO enzyme was previously detected in proteomes from both pathogenic and attenuated strains of M. hyopneumoniae (232 and J) [START_REF] Pinto | Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae[END_REF][START_REF] Pendarvis | Proteogenomic mapping of Mycoplasma hyopneumoniae virulent strain 232[END_REF], only the pathogenic strains tested in our study (7448 and 7422) were able to produce detectable amounts of hydrogen peroxide in Friis medium (Figure 1). To our knowledge, no other study up to now was able to show that M. hyopneumoniae strains were able to produce this toxic product in vitro [START_REF] Maes | Update on Mycoplasma hyopneumoniae infections in pigs: Knowledge gaps for improved disease control[END_REF]. We also show here that the production of hydrogen peroxide in the pathogenic strains of M. hyopneumoniae is dependent on the presence of glycerol (Figure 1B).

The metabolism of glycerol and the formation of hydrogen peroxide were described as essential for the cytotoxicity of lung pathogens M. mycoides subsp. mycoides [START_REF] Vilei | Genetic and biochemical characterization of glycerol uptake in Mycoplasma mycoides subsp. mycoides SC: its impact on H(2)O(2) production and virulence[END_REF] and M. pneumoniae [START_REF] Hames | Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae[END_REF]. Moreover, although both M. hyopneumoniae and M. flocculare can adhere to the cilia of tracheal epithelial cells in a similar way, only the adhesion of M. hyopneumoniae causes tissue damage [START_REF] Young | A tissue culture system to study respiratory ciliary epithelial adherence of selected swine mycoplasmas[END_REF].

We showed that the difference in enzyme activity was not related to the expression levels of glpO gene from the strains tested (Figure 2). We did not find any extreme differences in their aminoacid sequences either (Supplementary Figure S3). This could be an indication that either this enzyme undergoes posttranslational modifications in order to be active and/or the availability of the substrate (glycerol) intracellularly might be a limiting step for its activity. Posttranslational modifications have been extensively reported experimentally in several proteins of M. hyopneumoniae [START_REF] Djordjevic | Proteolytic processing of the Mycoplasma hyopneumoniae cilium adhesin[END_REF][START_REF] Burnett | P159 is a proteolytically processed, surface adhesin of Mycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryote cells[END_REF][START_REF] Pinto | Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel posttranslationally modified and antigenic proteins[END_REF][START_REF] Seymour | A processed multidomain Mycoplasma hyopneumoniae adhesin binds fibronectin, plasminogen, and swine respiratory cilia[END_REF][START_REF] Tacchi | Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae[END_REF]. From transcriptomic and proteomic literature data, we were not able to find any enlightening differences in this pathway between strains or species (Supplementary Table S7).

As for the availability of intracellular glycerol, in our previous metabolic models, we predicted differences in the metabolism of glycerol among the three Mycoplasma species (Supplementary Figure S6). While M. hyopneumoniae has five different ways of uptaking glycerol (dehydrogenation of glyceraldehyde, ABC transport of glycerol and glycerol-phosphate, import of glycerophosphoglycerol, and glycerophosphocholine), the other two species lack at least two reactions.

This might also limit the rate of production of hydrogen peroxide in each species.

In this way, the enhanced pathogenicity of M. hyopneumoniae over M. hyorhinis and M. flocculare may therefore also be due to hydrogen peroxide formation resulting from a higher uptake of glycerol as an energy source. Similarly, one reason that could partially explain why M. mycoides subsp. mycoides is highly pathogenic in comparison with the less pathogenic M. pneumoniae might be the greater intracellular availability of glycerol due to the presence of a specific and very efficient ABC transporter in M. mycoides subsp. mycoides.

Since the production of hydrogen peroxide was not reported as essential to the in vivo virulence of Mycoplasma gallisepticum [START_REF] Szczepanek | Hydrogen peroxide production from glycerol metabolism is dispensable for virulence of Mycoplasma gallisepticum in the tracheas of chickens[END_REF], more studies are needed to better understand the importance of this metabolism in M. hyopneumoniae. Moreover, future biochemical and functional studies are needed to prove that GlpO is indeed responsible for the activity proposed here and to check if the enzyme in attenuated strains/species is functional.

Myo-inositol uptake and catabolism

M. hyopneumoniae is the only Mycoplasma species with sequenced genome that has the genes for the catabolism of myo-inositol. Myo-inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes [START_REF] Gonzalez-Salgado | Myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei[END_REF]. Myo-inositol is also widespread in the bloodstream of mammalians [START_REF] Reynolds | Strategies for acquiring the phospholipid metabolite inositol in pathogenic bacteria, fungi and protozoa: making it and taking it[END_REF], which would make it a suitable energy source for bacteria in the extremely vascularized respiratory system. Previously, Mycoplasma iguanae was described to produce acid from inositol [START_REF] Brown | Mycoplasma iguanae sp. nov., from a green iguana (Iguana iguana) with vertebral disease[END_REF], but the methods used in that paper are not clear and there is no complete genome from this organism for us to draw any conclusions. Based on sequence homology, orthology, synteny and tridimensional analyses, we proposed a possible candidate for the missing enzyme IolJ in M. hyopneumoniae, namely a duplication of the fba gene from glycolysis. This functional divergence after duplication is particularly interesting in bacteria for which evolution was mostly driven by genome reduction. Another reported example of this event is the duplication of the trmFO gene in Mycoplasma capricolum and more recently in Mycoplasma bovis. The duplicated TrmFO in M. capricolum was reported to catalyze the methylation of 23S rRNA [START_REF] Lartigue | The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA[END_REF] while the duplicated copy in M. bovis has been described to act as a fibronectin-binding adhesin [START_REF] Guo | TrmFO, a Fibronectin-Binding Adhesin of Mycoplasma bovis[END_REF].

We showed here that M. hyopneumoniae was able to uptake marked myoinositol from a complex culture medium (Figure 5); in addition this was the only species that remained viable whenever myo-inositol was used as the primary energy source (Figure 6). From our metabolic model predictions [START_REF] Ferrarini | Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling[END_REF], the use of myo-inositol would be much more costly than the uptake and metabolism of glucose, which corroborates the small uptake of myo-inositol in Friis medium (glucose-rich) (Figure 5). This basal uptake of myo-inositol could also be an indication that this pathway is important not only for energetic yield. Supporting this idea, microarray studies on strain 232 showed that several genes (if not all) from the myo-inositol catabolism were differentially expressed during stress treatments: heat shock (downregulated) [START_REF] Madsen | Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays[END_REF], iron depletion (upregulated) [START_REF] Madsen | Transcriptional profiling of Mycoplasma hyopneumoniae during iron depletion using microarrays[END_REF], and norepinephrine (downregulated) [START_REF] Oneal | Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to norepinephrine[END_REF]. Moreover, a previous transcriptome profiling of M. hyopneumoniae [START_REF] Siqueira | Unravelling the transcriptome profile of the swine respiratory tract mycoplasmas[END_REF] showed that all genes from the myo-inositol catabolism were transcribed under normal culture conditions. Furthermore, three genes from the pathway (iolB, iolC and iolA) belonged to the list of the 20 genes with the highest number of transcript reads. Besides the transcription of these genes, proteomic studies of M. hyopneumoniae strains 232 [START_REF] Pendarvis | Proteogenomic mapping of Mycoplasma hyopneumoniae virulent strain 232[END_REF], 7422, 7448 and J [START_REF] Pinto | Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae[END_REF][START_REF] Reolon | Survey of surface proteins from the pathogenic Mycoplasma hyopneumoniae strain 7448 using a biotin cell surface labeling approach[END_REF] (Supplementary Table S7) showed that several enzymes from this pathway were present in normal culture conditions. Indeed, myo-inositol has been extensively reported in several organisms as a signaling molecule [START_REF] Downes | Myo-inositol metabolites as cellular signals[END_REF][START_REF] Gillaspy | The cellular language of myo-inositol signaling[END_REF]. Moreover, the myo-inositol catabolism has been experimentally described as a key pathway for competitive host nodulation in the plant symbiont and nitrogen-fixing bacterium Sinorhizobium meliloti [START_REF] Kohler | Inositol catabolism, a key pathway in Sinorhizobium meliloti for competitive host nodulation[END_REF]. Host nodulation is a specific symbiotic event between a host plant and a bacterium. Kohler and collaborators (2010) showed that whenever inositol catabolism is disrupted (by single gene knockouts from the inositol operon), the mutants are outcompeted by the wild type for nodule occupancy. This means that genes for the catabolism of inositol are required for a successful competition in this particular symbiosis. Moreover, the authors were not able to find a suitable candidate for the IolJ activity. In our case, we proposed that the activity of the missing enzyme IolJ is taken over by a duplication of fba. We were able to find a similar duplication (also not inside the myo-inositol cluster) in the genome of S. meliloti 1021 (SM_b21192 and SM_b20199, both annotated as fructosebisphosphate-aldolase, EC 4.1.2.13). This means that in at least one other symbiont that has the myo-inositol catabolism genes, there could exist a putative IolJ not close to the myo-inositol cluster, just as we proposed here.

Whether this entire pathway is functional in M. hyopneumoniae is yet to be tested and further experiments should take place to support this hypothesis. However, the ability of M. hyopneumoniae to persist longer in the swine lung if compared to the other two mycoplasmas might come from the fact that this species is able to uptake and process myo-inositol. Furthermore, the ability of M. hyopneumoniae to grow in diverse sites [START_REF] Carrou | Persistence of Mycoplasma hyopneumoniae in experimentally infected pigs after marbofloxacin treatment and detection of mutations in the parC gene[END_REF] if compared to M. flocculare might also be due to this specific trait.

Concluding remarks

It is important to remember that even though M. hyopneumoniae is considered highly pathogenic, the three Mycoplasma species studied here are widespread in pig populations and can easily be found in healthy hosts [START_REF] Fano | Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs[END_REF][START_REF] Pieters | An experimental model to evaluate Mycoplasma hyopneumoniae transmission from asymptomatic carriers to unvaccinated and vaccinated sentinel pigs[END_REF]. However, the main question permeating this fact is: what causes the switch from a nonpathogenic Mycoplasma community to a pathogenic one? And what makes some strains pathogenic while others inflict no harm to the host cells? Some strains of M. hyopneumoniae become less pathogenic in broth culture and, after serial passages, they lose their ability to produce gross pneumonia in pigs [START_REF] Whittlestone | Porcine mycoplasmas[END_REF]. In a proteomic study comparing strains 232 and J, researchers have described that the attenuated strain J switches its focus to metabolism and therefore has developed better capabilities to profit from the rich culture medium while the ability to infect host cells becomes less important so that adhesionrelated genes are downregulated [START_REF] Li | Proteomic comparative analysis of pathogenic strain 232 and avirulent strain J of Mycoplasma hyopneumoniae[END_REF]. This might be related to the fact that here we detected a higher production of ATP in this attenuated strain when compared to the pathogenic strains 7448 and 7422. Liu and collaborators [START_REF] Liu | Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic 168 strain and its high-passaged attenuated strain[END_REF] have investigated genetic variations between M. hyopneumoniae strains 168 and attenuated 168-L and found out that almost all reported Mycoplasma adhesins were affected by mutations. Tajima and Yagihashi [START_REF] Tajima | Interaction of Mycoplasma hyopneumoniae with the porcine respiratory epithelium as observed by electron microscopy[END_REF] reported that capsular polysaccharides from M. hyopneumoniae play a key role in the interaction between pathogen and host. Indeed in several bacterial species it has been reported that the amount of capsular polysaccharide is a major factor in their virulence [START_REF] Corbett | The role of microbial polysaccharides in hostpathogen interaction[END_REF] and it decreases significantly with in vitro passages [START_REF] Kasper | Capsular polysaccharides and lipopolysaccharides from two Bacteroides fragilis reference strains: chemical and immunochemical characterization[END_REF]. In this way, it is likely that the difference in pathogenicity between strains in M. hyopneumoniae does not solely depend on their metabolism, but also on their ability to adhere to the host.

A recent metagenomic analysis of community composition [START_REF] Siqueira | Microbiome overview in swine lungs[END_REF] has described that M. hyopneumoniae is by far the most prevalent species in both healthy and diseased hosts. The difficult isolation of Mycoplasma species from diseased lung extracts is due to the fact that, in culture, fast-growing bacteria will overcome the slow-growth of mycoplasmas [START_REF] Mckean | Evaluation of diagnostic procedures for detection of mycoplasmal pneumonia of swine[END_REF]. This means that, in vitro, the competition for an energy source between fast and slow-growing bacteria usually ends with an overpopulation of the fast growing ones. Given the fact that mycoplasmas survive for longer periods inside the host even in competition with other bacteria [START_REF] Fano | Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs[END_REF][START_REF] Overesch | Persistence of Mycoplasma hyopneumoniae sequence types in spite of a control program for enzootic pneumonia in pigs[END_REF], we must assume that other factors exist and are usually not mimicked in cell culture.

While M. hyopneumoniae might cause no harm, depending mostly on the environment, the characteristics of the host, and the composition of this dynamic lung microbiome, any unbalance in this system is probably capable of turning a non-pathogenic community into a pathogenic one. The final conclusion is that the disease is a multifactorial process depending on several elements that include intra-species mechanisms, community composition, host susceptibility and environmental factors. One possibility is that the competition with fast-growing species could result in a lower carbohydrate concentration and that M. hyopneumoniae might have to overcome this environmental starvation with the uptake of glycerol or myo-inositol. Since the uptake of myo-inositol does not lead to the production of any toxic metabolite, it is more interesting for its persistence in the long run. Other bacteria will strongly compete for glucose and other related carbohydrates, while M. hyopneumoniae will have the entire supply of myoinositol for itself. The uptake of glycerol as an energy source, on the other hand, will probably lead to the production of toxic hydrogen peroxide as reported in other Mycoplasma species. This toxic product combined with other toxins from the external bacteria in the system would most probably recruit immune system effectors. Since M. hyopneumoniae has efficient mechanisms of host evasion [START_REF] Fano | Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs[END_REF][START_REF] Maes | Update on Mycoplasma hyopneumoniae infections in pigs: Knowledge gaps for improved disease control[END_REF], the newly introduced and fast-growing bacteria might be eliminated faster and M. hyopneumoniae, in this way, would be able to persist longer than other species inside the host (as it is reported in vivo).

As mentioned before, virulence factors in Mycoplasma species cover a broader concept if compared to other species: they are genes not essential for in vitro conventional growth that are instead essential for optimal survival in vivo. From our M. hyopneumoniae metabolic models, neither the GlpO activity nor the uptake and metabolism of myo-inositol seem to be essential features for in vitro growth. However, we were able to show that they might be two metabolic traits important for the enhanced virulence of M. hyopneumoniae when compared to M. hyorhinis and M. flocculare and could be essential for its survival in vivo and directly affect its pathogenicity.

Experimental Procedures

Mycoplasma cultivation

We used the following strains for experimental validation: M. hyopneumoniae strains 7448, 7422 (field isolates) and J (ATCC 25934), M. hyorhinis ATCC 17981 and M. flocculare ATCC 27716. Cells were cultivated in Friis medium [START_REF] Friis | Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare a survey[END_REF] at 37 ∘ C for varying periods of time with gentle agitation in a roller drum.

Hydrogen peroxide detection

Hydrogen peroxide was detected in culture medium by the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen Cat. No A22188), according to the manufacturer's manual. M. hyopneumoniae, M. hyorhinis and M. flocculare were cultivated for 48 h in modified Friis medium (with no Phenol Red) and thereafter centrifuged. The supernatant was used for the hydrogen peroxide readings compared to a standard curve (Supplementary Figure S7). The medium without bacterial growth was used as negative control. We used biological and technical triplicates to infer the average amount of hydrogen peroxide produced, and the concentration was standardized based on the average number of cells from each culture. Statistical analyses were performed using GraphPad Prism 6 software by one-way ANOVA followed by Dunnett's multiple comparison test considering M. flocculare as a control (p < 0.05).

In order to determine if the hydrogen peroxide production was dependent on the glycerol metabolism, we used the Merckoquant Peroxide Test (Merck Cat. No 110011) with detection range of 0.5 to 25 μg of peroxide per mL of solution (as described in [START_REF] Hames | Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae[END_REF]). Fifteen mL of M. hyopneumoniae 7448 and 7422 strains were grown for 48 h in Friis medium, harvested by centrifugation at 3360 g and washed twice in the incubation buffer (67.7 mM HEPES pH 7.3, 140 mM NaCl, 7 mM MgCl 2 ). Cells were resuspended in 4 mL of incubation buffer and aliquots of 1 mL were incubated for 1 h at 37 ∘ C. To induce hydrogen peroxide production, either glycerol or glucose (final concentration 100 μM or 1 mM) was added to the cell suspension and samples were incubated at 37 ∘ C for additional 2 h. Hydrogen peroxide levels were measured using colorimetric strips according to the manufacturer's instructions. Aliquots without any added carbon source served as an incubation control. The statistical significance of the results was calculated using one-way ANOVA followed by Dunnett's multiple comparison test (p < 0.05). The results represent four biological replicates with at least two technical replicates each.

Mycoplasma cell count with flow cytometry

Mycoplasma cells cultivated for hydrogen peroxide detection were sedimented at 3360 g for 20 min at 4 ∘ C and washed three times with NaCl 0,9% (1x 3360 g for 20 min and 2x 3360 g for 4 min). Cells were resuspended in 1 mL of NaCl 0,9% and diluted 1:30 for flow cytometry readings in a Guava EasyCyte cytometer (Millipore, USA). Cells were characterized by side-angle scatter (SSC) and forward-angle scatter (FSC) in a four-decade logarithmic scale. Absolute cell counting was performed up to 5000 events and the samples were diluted until the cell concentration was below 500 cells/μL. The number of counts obtained was then converted to cells/mL.

Transcript levels of glpO with the use of real-time quantitative RT-PCR

Total RNA was isolated from 20 mL culture of M. hyopneumoniae strains 7448, 7422 and J grown at 37 ∘ C for 24 h. Cells were harvested by centrifugation at 3360 g for 15 min, resuspended in 1mL of TRizol (Invitrogen, USA) and processed according to the manufacturer's instructions followed by DNA digestion with 50 U of DNaseI (Fermentas, USA). Absence of DNA in the RNA preparations was monitored by PCR assays. The extracted RNA was analysed by gel electrophoresis and quantified with the Qubit TM system (Invitrogen, USA).

A first-strand cDNA synthesis reaction was conducted by adding 500 ng of total RNA to 500 ng of pd(N)6 random hexamer (Promega, USA) and 10 mM deoxynucleotide triphosphates. The mixture was heated for 65 ∘ C for 5 min and then incubated on ice for 5 min. First-strand buffer (Invitrogen, USA), 0.1 M dithiothreitol and 200 U M-MLV RT (Moloney Murine Leukemia Virus Reverse Transcriptase -Invitrogen, USA) were then added to a total volume of 20 μL. The reaction was incubated at 25 ∘ C for 10 min and at 37 ∘ C for 50 min followed by 15 min at 70 ∘ C for enzyme inactivation. A negative control was prepared in parallel, differing only by the absence of the RT enzyme. Quantitative PCR (qPCR) assay was performed using 1:2.5 cDNA as template and Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen, USA) with specific primers for glpO (5'GGTCGGGAACCTGCTAAAGC3' and 5'CCAGACGGAAACATCTTAGTTGG3') on StepOne Real-Time PCR Systems (Applied Biosystems, USA). The qPCR reactions were carried out at 90 ∘ C for 2 min and 95 ∘ C for 10 min followed by 40 cycles of 95 ∘ C for 15 s and 60 ∘ C for 1 min. A melting curve analysis was done to verify the specificity of the synthesized products and the absence of primer dimers. The amplification efficiency was calculated with the LinRegPCR software application [START_REF] Ruijter | Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data[END_REF]

Comparative modeling and protein-ligand interaction analysis of Fba and IolJ

The SWISS-MODEL server [START_REF] Schwede | SWISS-MODEL: An automated protein homology-modeling server[END_REF][START_REF] Biasini | SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information[END_REF] was used for template search and the comparative modeling for all Fba and IolJ proteins in this study. The best homology models were selected according to coverage, sequence identity, Global Model Quality Estimation (GMQE) and QMEAN statistical parameters [START_REF] Benkert | QMEAN server for protein model quality estimation[END_REF][START_REF] Benkert | Toward the estimation of the absolute quality of individual protein structure models[END_REF]. The Fba from M. hyopneumoniae along with IolJ and Fba from B.

subtilis were modeled using the crystal structure of fructose 1,6-bisphosphate aldolase from Bacillus anthracis in complex with 1,3-dihydroxyacetonephosphate (PDB 3Q94) while Fba-1 from M. hyopneumoniae was modeled using the fructose-1,6-bisphosphate aldolase from Helicobacter pylori in complex with phosphoglycolohydroxamic acid (PDB 3C52). Both selected templates have the same resolution range (2.30Å). Fba structures experimentally solved from E. coli [START_REF] Hall | The crystal structure of Escherichia coli class II fructose-1, 6-bisphosphate aldolase in complex with phosphoglycolohydroxamate reveals details of mechanism and specificity[END_REF] and G. intestinalis [START_REF] Galkin | Structural insights into the substrate binding and stereoselectivity of Giardia fructose-1,6-bisphosphate aldolase[END_REF] were used to include information about substrate binding in the active site. The DKGP and FBP ligands were drawn in the Avogadro version 1.1.1 [START_REF] Hanwell | Avogadro: an advanced semantic chemical editor, visualization, and analysis platform[END_REF] by editing the tagatose-1,6-biphosphate (TBP) molecule complexed with the Fba structure of G. intestinalis (PDB 3GAY). Each model was submitted to 500 steps of an energy minimization protocol using the universal force field (UFF). The DKGP and FBP molecules were inserted into the substrate binding sites of the acquisition models obtained by superposition of the models with the Fba structure of G. intestinalis.

Detection of marked myo-inositol through mass spectrometry

Sample preparation

All samples were filtered and concentrated with the use of Amicon Ultra 3 kDa (Merck Millipore Cat. No. UFC200324). After this step, samples were dried in a miVac sample concentrator (Genevac, Ipswich, UK) for approximately 45 min at 50 ∘ C. All samples were ressuspended in ultra pure water to a final concentration of 10 g/L and were subsequently submitted to mass spectrometry.

Mass spectrometry

Aqueous extracts of Mycoplasma sp. and commercial deuterated myo-inositol-1,2,3,4,5,6-d6 were analysed using an Accurate-Mass Q-TOF LCMS 6530 with LC 1290 Infinity system and Poroshell 120 Hilic column (3x100 mm, 2.7 μm) (Agilent Technologies, Santa Clara, USA). The extracts were dissolved in water (10 g/L) and injection volume was 3 μL. A binary mobile phase system (A: 0.4% formic acid in milliQ-water and B: acetonitrile) was pumped at a flow rate of 0.9 mL/min at the following gradient: 0-3.5 min, 90% B; 3.5-7 min, 90% to 0% B; 7-9.5 min, 0% B; 9.5-10 min 0% to 90% B; 10-15 min, 90% B (total run: 15 min).

MS and MS

Determination of cell viability of M. hyopneumoniae in myo-inositol defined medium

All available strains were grown in Friis medium at 37 ∘ C for 48 h, sedimented by centrifugation at 3360 g for 20 min at 4 ∘ C, washed twice with ice cold PBS and inoculated in glucose regular defined medium (described in [START_REF] Ferrarini | Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling[END_REF], supplemented with 5 g/L of succinate) or myo-inositol defined medium (regular defined medium depleted with glucose and glycerol and supplemented with 0.5 g/L of myoinositol). Viability of cells was measured by ATP production with live cells recovered after 8 h of growth in either media with a BacTiter-Glo TM Microbial Cell Viability Assay Kit (Promega, USA) according to the manufacturer's manual.

Luminescence was recorded in a SpectraMax MiniMax 300 Imaging Cytometer (Molecular Devices, USA) with an integration time of 0.5 s in an opaque-walled multiwell plate. Average ATP production was calculated with biological duplicates and technical triplicates. The ATP production of each strain was compared between regular defined medium and myo-inositol defined medium to determine the ratio of viable cells and to allow a comparison between strains. A 10-fold serial dilution of ATP was used as a standard curve (Supplementary Figure S8).

Statistical analyses were performed using GraphPad Prism 6 software by oneway ANOVA followed by Tukey's multiple comparison test (p < 0.05). Fig. 2 Expression levels of glpO gene in M. hyopneumoniae strains. We did not find any significant difference on the transcript levels of glpO from all tested strains. Bars show the average relative quantification normalized against unit mass (500 ng of total RNA) and replicate 2 from strain 7448 was used as the calibrator. Average expression levels were calculated with independent biological triplicates (p < 0.05). hyopneumoniae). In contrast, Fbas generally bear glycines in this position (for complete explanation see Supplementary Figures S3 andS4). While Fba-1 from M. hyopneumoniae resembles more the experimentally solved Fba enzymes from B. subtilis, E. coli and, G. intestinalis, the predicted structure of Fba from M.

hyopneumoniae is more similar to the IolJ structure from B. subtilis. hyorhinis ATCC 17981 (MHR). While there is no significant difference in the concentrations between MFL and MHR and the control medium (CTRL), both M.

hyopneumoniae strains seem to be able to uptake myo-inositol. B. We also collected two extra time points for MHP_7448 and CTRL: 8h and 24h of growth.

In all time points there is significant difference between residual marked myoinositol and the control medium. Data are presented as mean and standard deviation of 4 independent biological replicates. Asterisks indicate statistically significant differences in residual marked myo-inositol (*p < 0.05; **p < 0.01).

M. h y o p n e u mo n i a e M. h y o p n e u mo n i a e M. h y o p n e u mo n i a e M. h y o p n e u mo n i a e B. s u b t i l i s B. s u b t i l i s B. s u b t i l i s

1 3

 13 Comparative genomics of glpO from glycerol metabolism 1.2 Pathogenic M. hyopneumoniae strains produce hydrogen peroxide from glycerol 1.3 Levels of glpO transcripts do not differ from pathogenic to attenuated strains of M. hyopneumoniae 1.4 Enzymes for the uptake and catabolism of myo-inositol are specific to M. hyopneumoniae strains 1.5 M. hyopneumoniae is able to uptake myo-inositol from the culture Mycoplasma cell count with flow cytometry 3.4 Transcript levels of glpO with the use of real-time quantitative RT-PCR 3.5 Comparative modeling and protein-ligand interaction analysis of Fba and IolJ 3.6 Detection of marked myo-inositol through mass spectrometry 3.7 Determination of cell viability of M. hyopneumoniae in myo-inositol defined medium

  Acetonitrile and formic acid (Optima LC/MS Grade) were purchased from Fisher Scientific (Loughborough, UK). MilliQ water was obtained from a Direct-Q 5UV system (Merck Millipore, Billerica, Massachusetts, USA). Deuterated myoinositol-1,2,3,4,5,6-d6 was purchased from CIL (C/D/N Isotopes Inc. Cat No. D-3019, Canada).Cultivation in the presence of marked myo-inositolCells were cultivated in Friis medium supplemented with 0.25 g/L of deuterated myo-inositol-1,2,3,4,5,6-d6 (C/D/N Isotopes Inc. Cat No. D-3019). Cultures were interrupted after 8 h, 24 h and 48 h of cultivation for mass spectrometry analysis.

Fig. 1

 1 Fig.1Hydrogen peroxide production by swine mycoplasmas. A. In Friis medium after bacterial growth: Hydrogen peroxide was only detected in growth media from pathogenic strains (field isolates) of M. hyopneumoniae 7448 (MHP_7448) and 7422 (MHP_7422). Neither the attenuated strain J (MHP_J) nor the other species M. hyorhinis (MHR) and M. flocculare (MFL) produced detectable amounts of this toxic product. The concentration was also standardized based on the average number of cells from each culture. Data are presented as mean and standard deviation of three independent samples and statistical analysis was performed considering M. flocculare as a control strain (since it lacks the glpO gene). B. In the presence of different carbon sources: Pathogenic M. hyopneumoniae strains were used to test hydrogen peroxide production in incubation buffer supplemented with either glycerol or glucose after 2 h of incubation. Both strains were able to produce significant amounts of the

Fig. 3

 3 Fig. 3 Myo-inositol catabolism pathway in all M. hyopneumoniae strains and its transcriptional unit in M. hyopneumoniae strain 7448. Metabolites are depicted in dark green and enzymatic activities present in M. hyopneumoniae can be seen in pink. Metabolite abbreviations are as follows: MI (myo-inositol), 2KMI (2-ketomyo-inositol), THcHDO (3D-(3,5/4)-trihydroxycyclohexane-1,2-dione), 5DG (5deoxy-D-glucuronate), DKG (2-deoxy-5-dehydro-D-gluconate), DKGP (6phospho-5-dehydro-2-deoxy-D-gluconate), MSA (malonate semialdehyde), AcCoA (acetyl coenzyme-A), DHAP (dihydroxyacetone phosphate). EC 1.2.1.27:

Fig. 4

 4 Fig. 4 Substrate cavity prediction for Fba and Fba-1 from M. hyopneumoniae strain 7448. Cavities from the comparative models of Fba and Fba-1 from M. hyopneumoniae in comparison to the models constructed for Fba and IolJ from B. subtilis. The specificity for DKPG in IolJ seems to be strongly associated to the presence of a conserved arginine in position 'a' (R52 in Fba-1 from M.

Fig. 5

 5 Fig. 5 Deuterated myo-inositol-1,2,3,4,5,6-d6 uptake in complex medium. A. Comparison after 48 h of growth of M. hyopneumoniae J ATCC 25934 (MHP_J) and field isolate 7448 (MHP_7448), M. flocculare ATCC 27716 (MFL) and M.

Fig. 6

 6 Fig.6Viability of M. hyopneumoniae, M. hyorhinis and M. flocculare after 8 hours of incubation in myo-inositol defined medium. The viability of cells in myoinositol defined medium was measured by ATP production in comparison to inoculation in regular defined medium (glucose-containing medium). Data are represented as the ratio between ATP production in each media. There is a significant decrease of ATP production in M. hyorhinis and M. flocculare whereas at least 75% of the cells from M. hyopneumoniae remained viable after cultivation in the myo-inositol defined medium (***p < 0.001; ****P < 0.0001).

  . A relative quantification normalized against unit mass (500 ng of total RNA) was used to analyse the

	expression data with the equation:	( Ratio test calibrator /	) 2 CT  	, where
	CT CT  		CT	[80] and MHP_7448 (Replicate 2) was chosen as
	test		calibrator
	calibrator. Statistical analyses were performed using GraphPad Prism 6 software
	by one-way ANOVA followed by Tukey's multiple comparison test (p < 0.05).

  /MS spectra were obtained in negative mode, with the following conditions: nebulization gas (nitrogen) at 310 ∘ C, at a flow of 10 L/min and 40 psg pressure. The capillary tension was 3600 V and gave ionisation energy of 100 eV. In targeted MS/MS mode, collision energy was set at 18 eV. Acquisition range was m/z 50-500. MassHunter Qualitative Analysis Software (version B.07.00) was used for data analysis.

	Data analysis
	Deuterated myo-inositol-1,2,3,4,5,6-d6 was quantified in all aqueous extracts by
	HPLC-MS. For that, a calibration curve (based on peak area) of a commercial
	myo-inositol was performed from 0.001 g/L to 0.05 g/L in replicate (4 times during
	the batch analysis). Statistical analyses were performed using GraphPad Prism 6
	software. One-way ANOVA followed by Dunnett's multiple comparison test was
	used to test for differences in residual marked myo-inositol in culture after
	bacterial growth of all tested strains for 48 h (p < 0.05). A two-tailed unpaired t-
	test was used to compare the residual marked myo-inositol between M.
	hyopneumoniae 7448 and the control medium with two extra timepoints: 8 and
	24 h (p < 0.05).

Acknowledgments Authors' contributions

Acknowledgments

This work was supported by grants from CAPES-COFECUB 782/13 and Inria. MGF was granted post doctoral fellowship funded by the European Research Council under the European Community's Seventh Framework Programme (FP7 / 2007-2013)/ ERC grant agreement no. [247073]10. SGM was the recipient of a CAPES doctoral fellowship. DP was granted post doctoral fellowship funded by the European Union Framework Program 7, Project BacHbERRY number FP7-613793. JFRB is a recipient of a CAPES postdoctoral fellowship. The mass spectrometry analysis was carried out in the Centre d'Etude des Substances Naturelles at the University of Lyon.

Competing interests

Authors' contributions

MGF, SGM, MFS and AZ conceived and designed the work. MGF and SGM performed most of the experimental work. DP, GM and GC collaborated in the mass spectrometry experiments and analysis. JFB performed tridimensional analysis of proteins. All authors collaborated in the analysis of all data. MGF and SGM wrote the manuscript with inputs from the other authors. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests. Table S1: Cytometry cell counts and replicate readings for the calculation of hydrogen peroxide production. Table S2: Replicate readings of real-time RT qPCR for glpO transcript relative expression. Table S3: Gene locus tag for the genes from the uptake and metabolism of myo-inositol in M. hyopneumoniae strains. Table S4: Comparative modeling summary. Table S5: Average peak surface for marked myo-inositol from mass spectrometry experiments. Table S6: ATP production replicates and average for each sample. Table S7: Literature experimental data available for genes important for the glycerol and myo-inositol metabolism.

Supplementary material

File S1: Statistical analyses results.