PhD Susanne Fuchs
email: fuchs@zas.gwz-berlin.de

Jana Brunner

Anke Busler

Temporal and spatial aspects concerning the realisations of the voicing contrast in German alveolar and postalveolar fricatives

Keywords: Voicing contrast, devoicing, sibilants production, spatio-temporal aspects, laryngeal-oral coordination

This study investigates the phonetic realisations of voicing contrast in alveolar and postalveolar fricatives production in different word positions in order to understand the temporal and spatial production strategies used in the control of voicing and frication, and to provide a frame of reference for speech therapy despite the inter-speaker variation. Seven native speakers of German, originally coming from various regions, participated in the experiment. Acoustic signals were recorded onto DAT, and tongue palate contact patterns were recorded by means of electropalatography (EPG). The temporal parameters were measured using the acoustic signals and the spatial parameters were measured based on the EPG data. The corpus included real words with /s, z, S, Z/ occurring at word initial, medial and final positions. Temporal results showed that differences in the overall frication duration for voicing contrast occur at almost all positions (with longer duration for voiceless phonemes). However, voicing during the frication interval was a less reliable discriminator, particularly for Southern German speakers and at word final position. We found a positive correlation between the relative voicing duration and the amount of tongue palate contact for subjects who produced voicing. Especially for the postalveolars, voicing also coincides with more front articulation. Results are discussed with respect to laryngeal-oral co-ordination and aerodynamics.

Introduction

Alveolar and postalveolar fricatives, the sounds which will be studied here, present a particular challenge since firstly, they are difficult to acquire and susceptible to distortions, and secondly, the mechanisms underlying the production of the voicing contrast in fricatives, frication and voicing, are to some extent contradictory. Details of these mechanisms are rarely investigated and may therefore have led to divergent articulatory findings. The following sections provide a theoretical background for the facts mentioned above.

Distortions of alveolar and postalveolar fricatives

Alveolar and postalveolar fricatives are sounds which are very often distorted, especially by patients who suffer from sensori-motor coordination problems, hearing impairments, or anatomical abnormalities such as cleft palate (e.g., [START_REF] Boomer | Slips of the tongue[END_REF]Dagenais & Critz-Crosby, 1991;[START_REF] Fletcher | Teaching consonants to profoundly hearing-impaired speakers using palatometry[END_REF][START_REF] Gibbon | Abnormal patterns of tongue-palate contact in the speech of individuals with cleft palate[END_REF]. [START_REF] Fletcher | Teaching consonants to profoundly hearing-impaired speakers using palatometry[END_REF] described the treatment of profoundly hearing-impaired children using electropalatography (EPG) and they showed that learning /z/ was the most difficult task in comparison to stops (least difficult) or voiceless fricatives. They suggested that this finding could be due to the different physiological complexity of these sounds with /z/ being the most complex phoneme. This suggestion is in agreement with results reported by [START_REF] Fuchs | What role does the palate play in speech motor control? Insights from tongue kinematics for German alveolar obstruents[END_REF], who found evidence for two different motor control strategies for normal productions of alveolar stops (/t/) and fricatives (/z/). For alveolar stops, the tongue is not controlled via a fine positioning at the alveolar ridge, but the movement is planned towards a target above the alveolus, resulting in a collision of the tongue at this location (for the idea of this control strategy, see also [START_REF] Löfqvist | Lip and jaw kinematics in bilabial stop consonant production[END_REF]. Hence, the palate is used as a spatial reference which automatically blocks the tongue's movement. In terms of stability and simplicity, this control strategy seems to be extremely efficient in comparison to positioning precisely the lateral margins of the tongue at the palate, which is a more difficult control strategy (probably involving feedback information) used in the production of alveolar fricatives.

Conventional treatment of phonetic disorders consists of various steps. It starts with a verbal description of the articulation of the relevant sounds by the speech-language pathologist. It then continues with a process involving the perceptual discrimination between the distorted production and the target sound by the client, followed by a learning process of the appropriate pronunciation due to imitation of single versus larger speech units by the client. The additional use of visual feedback via EPG has been proven to be a successful tool in treating articulation disorders in different clinical populations, for instance, children with cleft palate [START_REF] Gibbon | Abnormal patterns of tongue-palate contact in the speech of individuals with cleft palate[END_REF][START_REF] Michi | Role of visual feedback treatment for defective /s/ sounds in patients with cleft palate[END_REF], children with persisting speech difficulties without any known organic aetiology [START_REF] Carter | EPG therapy for children with longstanding speech disorders: Predictions and outcomes[END_REF], or individuals with hearing-impairment [START_REF] Fletcher | Teaching consonants to profoundly hearing-impaired speakers using palatometry[END_REF]. In particular, EPG speeds up the learning process of correct /s/ pronunciation in comparison to conventional speech therapy without any visual feedback [START_REF] Michi | Role of visual feedback treatment for defective /s/ sounds in patients with cleft palate[END_REF].

One of the general difficulties for speech therapy consists of a clear definition of 'normal' and 'pathological' speech. Roberts, McLeod and Sita (2002) searched the EPG literature for normal and impaired /s/ and /z/ productions and divided the results for impaired alveolar fricatives into the following categories: higher overall amount of tongue-palate contacts, higher amount of contacts in the palatal region, lateral gaps or fewer tongue palatal contacts in comparison to the normal (for a case study on impaired /s/, see also [START_REF] Gibbon | Articulatory description and treatment of "lateral /s/" using electropalatography: A case study[END_REF]. Impaired alveolar fricative productions were manifold and sometimes contradictory, hence, no general pattern emerged. Slight asymmetries in the lateral contacts were no sign for impaired alveolar fricative productions and were frequently found in the normal patterns (see also [START_REF] Hamlet | Articulatory asymmetries [Letter to the Editor[END_REF], for EPG patterns produced by normal speakers).

To summarise, alveolar and postalveolar fricatives are sounds which are frequently distorted by patients who have speech disorders due to different aetiologies. Distortions are probably caused by the more difficult speech production mechanisms of these sounds, in comparison to others such as alveolar stops. EPG is useful in speech therapy to improve the speed of the learning process of correct articulation. However, no unique pattern emerges when EPG data from normal and abnormal alveolar fricatives are compared.

The voicing contrast in fricatives

Although various studies have been carried out to investigate the voicing contrast in stops, detailed analyses of articulatory mechanisms involved in the voicing contrast of fricatives are rather rare, and often differ in their interpretation.

Contradictory control strategies: Voicing and frication

It is well known that voicing and frication demand different control strategies concerning the intraoral and subglottal pressure conditions in the vocal tract. Theoretically, voicing requires an adducted glottis and a transglottal pressure difference, with the pressure below the glottis (hereafter subglottal pressure) higher than the pressure in the oral cavity (hereafter intraoral pressure). This transglottal pressure difference is observed in an open vocal tract configuration, such as occurs during open vowel production. However, during the production of obstruents, the intraoral pressure rises before full articulatory closure or constriction is reached [START_REF] Müller | Variations in the supraglottal air pressure waveform and their articulatory interpretation[END_REF]. The intraoral pressure may rise to such an extent that the transglottal pressure difference necessary for voicing cannot be maintained, thus, the phonologically voiced obstruents become devoiced or partly devoiced. This is true unless other mechanisms such as cavity enlargement are realised, which aim to maintain the transglottal pressure difference (for an overview on cavity enlargement strategies in stop production, see [START_REF] Fuchs | Articulatory correlates of the voicing contrast in alveolar obstruent production in German[END_REF]. Cavity enlargement has primarily been discussed with respect to stops production. Experimental evidence for its involvement in fricatives production has not been reported (except [START_REF] Narayanan | An articulatory study of fricative consonants using magnetic resonance imaging[END_REF]. It is possible to employ the strategy of cavity enlargement to maintain voicing during the production of obstruents by shifting the place of closure or constriction further to the front.

The production of frication requires an abducted glottis, a significant pressure drop across the oral constriction, and a high amount of airflow. An abducted glottis leads to a high amount of airflow. The pressure drop is created with a higher intraoral than atmospheric pressure by means of a small constriction and a larger cross-sectional area of the glottal constriction than the supraglottal constriction [START_REF] Stevens | Airflow and turbulence noise for fricative and stop consonants: Static considerations[END_REF]. Airflow usually becomes turbulent when leaving the narrow channel and/or hitting an obstacle in the vicinity of the air stream. The teeth are the obstacle during the production of alveolar and postalveolar fricatives [START_REF] Shadle | Articulatory-acoustic relationships in fricative consonants[END_REF][START_REF] Shadle | The effect of geometry on source mechanisms of fricative consonants[END_REF].

When producing voiced fricatives, a very precise laryngeal-oral adjustment is assumed in order to balance the two contradictory mechanisms: a higher intraoral than atmospheric pressure for frication and a lower intraoral than subglottal pressure for voicing. [START_REF] Mawass | Synthèse articulatoire des consonnes fricatives du français[END_REF] studied the area of the supraglottal and glottal constriction for a given subglottal pressure by means of a model. He discussed about the region where both voicing and frication can be realised and this region is indeed very narrow. If the pressure balance between frication and voicing is not achieved, voicing is more likely to disappear than frication, resulting in devoicing of phonologically voiced fricatives rather than the loss of frication [START_REF] Smith | The devoicing of /z/ in American English: Effects of local and prosodic context[END_REF]. The phonological terms, voiced and voiceless, are misleading to some extent, since phonologically voiced fricatives can be partially or totally devoiced. Hence, we will hereafter separate especially between 'phonologically voiced' and its actual realisation.

Inconsistent results from the literature

EPG studies which have investigated voicing contrast in normal speech production showed contrasting findings. On one hand, there are studies reporting no significant differences in EPG patterns between /s/ and /z/ or /S/ and /Z/ (e.g., [START_REF] Fletcher | Palatometric specification of stop, affricate, and sibilant sounds[END_REF][START_REF] Tabain | Variability in fricative production and spectra: Implications for the hyperand hypo-and quantal theories of speech production[END_REF], which suggest that voicing contrast in fricatives is only made at the laryngeal level, rather than the supralaryngeal level. On the other hand, the latest EPG studies on /s/ and /z/ production in English [START_REF] Mcleod | Tongue/palate contact for the production of /s/ and /z[END_REF] and Hindi [START_REF] Dixit | Articulatory characteristics of fricatives and affricates in Hindi: An electropalatographic study[END_REF] showed a greater percentage of anterior tongue palate contact patterns and a smaller groove width for the voiced segments. [START_REF] Dixit | Articulatory characteristics of fricatives and affricates in Hindi: An electropalatographic study[END_REF] suggested that the greater amount of EPG contact during the production of phonologically voiced fricatives could be a consequence of laryngeal action. During /s/ realisation, the glottis is open which guarantees the high amount of airflow necessary for the production of frication. In contrast, the glottis is adducted or slightly abducted when producing /z/. This impedes the airflow and the rise of intraoral pressure, causing insufficient intraoral pressure to produce frication. In such circumstances, a narrower groove resulting in more tongue palate contact would be a strategy to guarantee the pressure drop across the oral constriction, and thereby supports the creation of turbulent noise.

The controversial results reported in previous EPG studies could be due to the fact that phonologically voiced fricatives are often realised as partially devoiced or voiceless [START_REF] Smith | The devoicing of /z/ in American English: Effects of local and prosodic context[END_REF], and therefore the results become more similar to those of the phonologically voiceless phonemes. On the other hand, there are also certain environments or prosodic conditions in which phonologically voiced fricatives are realised as voiced and differ from voiceless.

Hence, taking into account the actual realisation of voicing during frication in phonologically voiced and voiceless phonemes and their EPG data should enhance the understanding of the voicing contrast and its control strategies.

The aims of this study are, firstly, to investigate the realisations of voicing contrast in alveolar and postalveolar fricatives with respect to different word positions, and secondly, to understand the possible control strategies used for the simultaneous production of voicing and frication, two contradictory mechanisms with respect to aerodynamic requirements. The third aim is to provide a frame of reference for normal speech production. To achieve these, temporal parameters were calculated by means of the analysis of acoustic signals, and spatial parameters were calculated using EPG.

Method

German was chosen because, to our knowledge, there is a gap in the knowledge on voicing contrast in alveolar and postalveolar fricatives [START_REF] Jessen | Phonetics and phonology of tense and lax obstruents in German[END_REF], and additionally, German is known for the devoicing of phonologically voiced tokens in word initial and word final positions.

Stimuli

The German sound inventory consists of the following fricatives: /f, v, s, z, S, Z, C, x, ", X, h/. The present study focuses on the alveolar and postalveolar fricatives /s, z, S, Z/ and their peculiarities since the other fricatives are either subject to inventory restrictions with respect to vowel environment or they do not have a phonemic counterpart. The labiodentals are not included because their place of constriction cannot be captured by means of EPG.

Concerning alveolar fricatives, a few minimal pairs can be found in intervocalic word medial position. [START_REF] Mangold | Zur Frage der Opposition /s/: /z/ im Deutschen[END_REF] counted 56 minimal pairs in the German lexicon including words with inflection, but he found only 11 real word pairs, for instance: Muse "muse" [:mu˘z´] vs. Muße "leisure" [:mu˘s´]. All other pairs also involve a difference regarding the preceding vowel context. Phonologically voiced alveolar fricatives tend to coincide with preceding tense vowels or diphthongs and phonologically voiceless fricatives with lax vowels, for example, Risse "rifts" [:"Is´] vs. Riese "giant" [:"i˘z´]. In addition, Mangold found twice as many words with /i˘z/ than with /i˘s/.

Only phonologically voiced alveolar fricatives exist at word initial position, except in the non-native vocabularies originated from English, Latin, Greek, and French [START_REF] Mangold | Zur Frage der Opposition /s/: /z/ im Deutschen[END_REF]. This restricted inventory is surprising since word initial stressed positions are prosodically strong positions and should coincide with longer segment durations and more extreme articulatory patterns than weak positions [START_REF] Keating | Domain initial articulatory strengthening in four languages[END_REF]. These temporal and spatial parameters are however known to facilitate devoicing. That is, voiceless phonemes are often longer than voiced ones, and extreme articulatory patterns such as increased tongue palate contact rather speak for a smaller oral constriction and therefore devoicing. It is unclear why only phonologically voiced alveolars exist in such a strong position. The latest work based on the PhonDat2 database, a corpus of hand-labelled utterances of 16 speakers of different origins in Germany [START_REF] Heid | Phonetische variation: Untersuchungen anhand des PhonDat2-Korpus [Phonetic variation -examinations based on the PhonDat2 corpus[END_REF], provided quantitative results for regional variations with respect to devoicing of initial /z/. Southern German speakers mostly devoice initial /z/ in contrast to Northern German speakers.

Contrary to the alveolar fricatives, only voiceless fricatives exist in initial position for postalveolars, except the non-native vocabulary, for example, Genie "genius" [ZAE:ni˘]. To round off the picture, the phonological rule of final devoicing applies at word final position (for an overview, see [START_REF] Brockhaus | Final devoicing in the phonology of German[END_REF]. The phonological rule is motivated by neutralisation of the voicing contrast in word-, morpheme-or syllable-final position, that is, phonologically voiced obstruents become voiceless.

To investigate the voicing contrast in German sibilants, a corpus following the German peculiarities in the different word positions was assembled. Some non-native vocabularies were included, particularly in those cases where a phonemic counterpart is missing in the German lexicon. Table 1 displays the selected target words. It should be noted that word pairs like Nase versus nasse also involve a tense-lax difference in the preceding stressed vowel, a fact which could have influenced the amount of tongue palate contact during the fricative production, since tense vowels are known to show more peripheral articulation than lax vowels in stressed position. However, the German /a/ is an exception, where there are only quantitative (temporal), but no qualitative differences, at least for Northern German speakers [START_REF] Kohler | Einführung in die Phonetik des Deutschen[END_REF].

Insert table 1 around here

All target words were embedded in the carrier phrase, Sage __ bitte "Say __ please" and they were repeated ten times in randomised order. This resulted in approximately 190 tokens for each subject (1326 tokens for all speakers, four tokens were missing). The full set of speech materials also includes words where stops are located at different word positions and this will be investigated in future work.

Speakers

In order to investigate temporal and spatial characteristics of alveolar and postalveolar fricatives production in German, the speech of seven native speakers, four male speakers (BM, DM, JM, RM) and three female speakers (CF, JF, SF), were recorded by means of electropalatography (Reading system, EPG3). Speech signals were simultaneously recorded on a Digital Audio Tape (DAT). Sampling frequencies were 48 kHz for the acoustic data and 100 Hz for EPG. All of the participants in this study are colleagues from the phonetics laboratory at the Centre for General Linguistics (ZAS) in Berlin, who have no known history of speech, language or hearing impairment, except for subject BM, who has a velopharyngeal dysfunction and a left-sided hearing impairment (both due to a traumatic accident). However, his daily speech does not sound impaired as judged by the colleagues in the lab and by his former students. Additionally, he was frequently a subject in electromagnetic articulography recordings, and his tongue movements did not show any peculiarities in comparison to other subjects.

Although all the speakers have lived in Berlin for at least 8 years, they grew up in different regions of Germany: JM and RM stem from the Mecklenburg area, the Northern part of former East Germany (close to Rostock); BM and CF are from Munich (Bavaria) in Southern Germany; SF grew up in the South of East Germany (Saxonia, close to the Czech border); DM is originally from Berlin and JF lived in Berlin most of her life.

Measurements and procedures

Temporal measures and procedures

For the acoustic analysis, the following time points were labelled using Praat (version 4.4.04) [START_REF] Boersma | Praat: Doing phonetics by computer (Version 4.4.04[END_REF]):

•

Frication onset (fric_on): the beginning of high frequency noise in the spectrogram;

• Voicing offset (voic_off): the voicing offset during the frication interval (the pitch tracker in Praat was used as a reference, with manual correction in case of faulty automatic calculations);

• Frication offset (fric_off): the end of high frequency noise in the spectrogram;

• F2 onset (f2_on): the beginning of the second formant (formants may be found throughout the whole frication segment in voiced intervocalic fricatives. In such cases, the increase in intensity and the increase in the acoustic envelope were taken into account as well); and

• F2 offset (f2_off): the end of the second formant (only the stressed vowels were considered).

Based on these landmarks, the following segment durations were calculated:

Frication duration: fricdur = fric_off -fric_on (1)

Voicing duration: voicdur = voic_off -fric_on

Relative voicing duration: voicdur_rel = voicdur *100/fric_dur (3)

Vowel duration of the stressed vowel: voweldur = f2_off -f2_on.

(4)

EPG measurements and procedures

The EPG data were imported into Matlab. The following parameters were measured within the acoustically defined frication interval:

• Percent of contact (PC): percent of contact over the whole palate

•

Percent of anterior contact (ANT): percent of contact in the first 4 rows

• Centre of gravity (COG) index: weighted index in the front-back dimension, with higher values corresponding to more anterior articulation [START_REF] Hardcastle | EPG data reduction methods and their implications for studies of lingual coarticulation[END_REF].

The ANT is a reasonable measure to account for the voicing contrast in alveolar fricatives.

However, it is not a reliable measure for the description of the postalveolar contrast, since some of the speakers in the present study realised the oral constriction of these sounds in a more posterior position than the ANT can capture. Hence, PC was chosen for the analysis of the postalveolars. The COG index was included as well since forward versus backward movement is another strategy to manipulate the aerodynamic conditions in the oral cavity.

Within a given frication interval, we averaged the corresponding ANT or PC or COG values in each repetition. The relative frequency of contact was calculated for the ten repetitions at the first time point in the frication interval where the ANT (for alveolars) or PC (for postalveolars) reached its maximum constriction. This time point was assumed to be the articulatory target.

SPSS 11.5 software package [START_REF] Jannsen | Statistische Datenanalyse mit SPSS für Windows[END_REF] was used for statistical analysis.

Results

Temporal results: Voicing and frication duration

Figure 1 shows the averaged temporal results split by phoneme, word position, and speaker. The complete bars correspond to the frication interval of the relevant phoneme ordered by word position. The voiced interval is marked in grey and the voiceless interval is displayed in black.

Insert figure 1 around here One of the major differences between phonologically voiced and voiceless phonemes is the overall duration of the frication interval, with longer durations for the voiceless even at the neutralisation position (word final, except for SF). However, if the differences in voicing duration are as pronounced as in /S/ and /Z/ at word medial position for RM, SF, and JM, the overall frication duration can be similar for the contrast pair. Additionally, figure 1 clearly displays two results: first, the actual realisation of voicing in the frication interval depends on word position and second, it depends on the speaker. The most likely position where a 'true'

voicing contrast is produced is the word medial position. The position where voicing disappears quickly or where it is not realised is the word final position. Results for the word initial position varied according to the speaker. BM, CF and SF do not produce any obvious differences in voicing duration between the phonologically voiced and voiceless fricatives.

These three speakers originally stem from Southern parts of Germany. For the two Bavarians, BM and CF, it is even the case that the phonological voicing contrast is never realised on the basis of differences in voicing duration. For SF, differences in voicing duration are still observed in the word medial position. The other subjects, who originally stem from the Northern parts of Germany, show a considerably longer voicing duration for phonologically voiced phonemes at word initial and medial positions than for the voiceless cognates.

However, the size of the samples in the present study is too small to test the difference between speakers with respect to their regional origin and further work is necessary to provide data on regional and dialectal variations.

Results for spatial parameters: ANT, PC and COG

Figures 2 and4 show the results for the spatial parameters of the voicing contrast measured with EPG. For visualisation purposes, the COG index and its standard deviation for each of the speakers was multiplied by 9. Figure 2 displays the results of ANT and COG index for the alveolars, and figure 4 shows the results of PC and COG index for the postalveolars.

Insert Figure 2 around here

In general, for the alveolars, the differences in ANT are most pronounced at word initial position (BP, DM, JF, JM, RM, SF with p < 0.004 for all subjects), followed by word medial position (p < 0.018 for DM and JF; p > 0.001 for JM, RM, and SF). At word final position, the differences are not significant in six out of seven speakers (except JM).

Generally, if differences between phonologically voiced and voiceless alveolars occur, then more EPG contacts are on average realised in phonologically voiced alveolar fricatives than in their voiceless counterparts. The differences are very subtle and often correspond to one or two electrodes. However, as mentioned in the introduction, the glottal constriction area has to be larger than the oral constriction to create a pressure drop across the oral constriction [START_REF] Stevens | Airflow and turbulence noise for fricative and stop consonants: Static considerations[END_REF]. This holds true for voiceless fricatives, where the glottis is open. Indeed, a very narrow oral constriction was found for these sounds. In phonologically voiced phonemes, an even smaller constriction width was produced which was verified on the basis of the EPG frequency plot. An example is provided in figure 3, for subject DP. The area where none of the EPG electrodes ever had contact is surrounded by a black line. In this particular case, a clear difference between the size of the constriction channels can be seen, with a smaller constriction width for /z/ than for /s/. Frequency of contact was also taken into account.

Insert Figure 3 around here Differences in constriction size may also be tiny, since the actual distance between the electrode's placement in the artificial palate (Reading system) is on average 2.89 mm in the first two anterior rows and 3.13 mm in the third and fourth row (in Fuchs, Brunner, & Busler, in preparation; data are based on 20 EPG palates including the subjects of this study).

Descriptions of the constriction width in alveolar fricative production varies from 6-12 mm [START_REF] Ladefoged | The sounds of the world's languages[END_REF]. Differences in the constriction width of one or two electrodes are therefore large in terms of the creation of a pressure drop.

The results of the COG differences in alveolars appear similar to those of the ANT differences, but are less pronounced. The voicing contrast in the production of postalveolars as shown in figure 4 involves significant differences in front versus back articulation, that is, phonologically voiced tokens are realised further to the front than their voiceless counterparts (p < 0.001 for DM, JF, JM, and RM at medial position; JM and DM at word final position).

Insert Figure 4 around here

The results also show greater PC values in the phonologically voiced phonemes than the voiceless ones, but the differences are smaller. A contrasting result, with more contacts for the voiceless phonemes, was found in CF and SF.

Relation between temporal and spatial aspects

As mentioned earlier, previous findings (e.g., [START_REF] Dixit | Articulatory characteristics of fricatives and affricates in Hindi: An electropalatographic study[END_REF][START_REF] Fletcher | Palatometric specification of stop, affricate, and sibilant sounds[END_REF][START_REF] Mcleod | Tongue/palate contact for the production of /s/ and /z[END_REF][START_REF] Tabain | Variability in fricative production and spectra: Implications for the hyperand hypo-and quantal theories of speech production[END_REF] differed with respect to whether they found a supralaryngeal involvement in terms of tongue palate contact in the voicing contrast of fricatives or not. None of these studies considered the actual phonetic realisations of the phonologically voiced phonemes. They can become devoiced and thereby similar to the voiceless. To take this factor into account, the correlations between the following parameter were evaluated: ANT and relative voicing duration; COG index and the relative voicing duration for the alveolar fricatives; PC and relative voicing duration; COG index and relative voicing duration for the postalveolars. All phonologically voiced and voiceless fricatives either alveolar or postalveolar were pooled together in order to base the analysis on the phonetic realisation, instead of the phonological terms. Note that, first, the Spearman Rho correlation, which does not assume normally distributed variables, was used. The relative voicing duration was bimodally distributed for all subjects except BM and CF. The bimodal distribution was not a result of pooling phonologically voiced and voiceless together. It was due to the fact that a large number of data were fully voiced and the rest were only partially voiced. Second, since word position (prosody) may also affects the segment's duration, the relative voicing duration, which is a normalised voicing duration, was used for calculating the correlations. Table 2 depicts the results split by speaker.

Insert table 2 around here

In general, for all the subjects who produced voicing, significant positive correlations were found, that is, longer relative voicing duration coincides with a greater percentage of either ANT for the alveolars (except BM, CF) or PC for the postalveolars (except BM, CF, DM). Correlation coefficients may be weak in some cases since the differences in tongue palate contact, although significant, are very subtle and correspond quite often to contact differences of one to three electrodes. However, in terms of oral constriction, such difference may have a major effect on the width of the air channel and therefore on the production of turbulent noise.

Interestingly, for DM and RM, the relative voicing duration correlates to a somewhat greater extent with the COG for the alveolars. In the postalveolars, DM showed an insignificant correlation between the relative voicing duration and the PC, whereas there was a significant correlation between the COG index and relative voicing duration. Since in these cases a more frontal articulation corresponds to longer relative voicing, this result may rather speak for a strategy to enlarge the oral cavity and decrease intraoral pressure in order to facilitate voicing than for a mechanism to increase intraoral air pressure and the amount of airflow.

The data were splitted with respect to word position and the correlations between these parameters were evaluated again: at word initial position, all speakers showed a positive significant correlations ranging from 0.324 to 0.723 for the alveolars (except SF who showed insignificant correlation, and CF with negative correlations). At word medial position, the correlations are less pronounced and occur only for JM, RM, and SF (PC-voicdur_rel) and JF, JM, RM, and SF (COG-voicdur_rel). This effect may be explained in terms of the tense-lax differences in the preceding vowel which often coincide with differences in the phonological voicing status. No significant correlation was found for the word final position (position for neutralisation).

For the postalveolars, we could only consider the word medial and final positions. The word medial position is clearly the one where a strong positive relation between temporal and spatial parameters exist, except for BM, CF, SF for the COG, and RW for the PC. For all other speakers, significant correlation coefficients between 0.532 and 0.845 were found. All subjects showed greater correlations between COG and voicdur_rel than PC and voicdur_rel.

Voicing coincides with a more frontal articulation and more tongue palate contact patterns for the postalveolars. The strength of the effect in the postalveolars can be explained by the fact that these sounds are not limited in their degree of freedom, whereas tongue movement in the alveolar production is limited due to the teeth. Additionally, the more posterior the overall constriction, the higher the intraoral pressure and the more likely the devoicing of phonologically voiced tokens [START_REF] Ohala | The origin of sound patterns in vocal tract constraints[END_REF]. Placing the tongue further to the front may be a necessary strategy to maintain the transglottal pressure difference and thus, voicing.

At word final position (position for final devoicing), six out of seven subjects show no significant correlation, only DM showed a strong correlation between COG and voicdur_rel.

Summary and conclusion

By means of acoustics and electropalatography, the present study investigated the temporal and spatial characteristics of voicing contrast in fricative production at three different word positions. Seven native speakers coming originally from different regions in Germany were recorded. This study could be seen as a step towards the understanding of possible control strategies for the realisation of both voicing and frication, two rather contradictory mechanisms with respect to aerodynamic requirements. This study also provides a frame of reference for speech therapy, since alveolar fricatives are frequently distorted in particular.

The results of the temporal parameters revealed that, for voicing contrast, the differences in overall frication duration were generally more stable than the differences in voicing duration, since Southern German speakers realise all phonologically voiced tokens in the main part of the frication interval as voiceless, but the voiced phonemes are still shorter in overall duration in comparison to the voiceless ones. Difference in frication duration were also found for the word final position (position for neutralisation), but the extent of difference was smaller and it was not observed in every subject. Considering spatial aspects, one of the major outcomes of this study is the higher amount of anterior contact in phonologically voiced than in voiceless alveolars. These results are only found in those speakers who realise voicing but not for those who devoice. The greater amount of contact is attributed to a narrower medial groove. For postalveolars, similar but less pronounced differences have been shown for the PC. An additional and even stronger characteristic was that speakers who realise a true voicing contrast place their tongue further to the front (anterior) in the voiced postalveolars than in the voiceless. This mechanism has been interpreted with respect to the maintenance of voicing.

We conclude that the voicing contrast involves an appropriate laryngeal-oral coordination and is not only realised due to differences at the laryngeal level. The present EPG results are in agreement with [START_REF] Mcleod | Tongue/palate contact for the production of /s/ and /z[END_REF] and [START_REF] Dixit | Articulatory characteristics of fricatives and affricates in Hindi: An electropalatographic study[END_REF]. In addition, for the alveolars we found evidence that the relative voicing duration and the percent of anterior contacts correlate, that is the longer relative voicing duration, the more anterior contacts are produced. We conclude that for alveolars, building a narrower channel where air is passing through is an appropriate strategy to create a pressure drop, increase the airflow, and thereby to realise turbulent noise even when the glottis is only slightly open or closed [START_REF] Dixit | Articulatory characteristics of fricatives and affricates in Hindi: An electropalatographic study[END_REF]. The realisation of such a small constriction channel should be particularly sensitive to distortions and may also be one of the reasons why patients have the most difficulty in producing these sounds [START_REF] Fletcher | Teaching consonants to profoundly hearing-impaired speakers using palatometry[END_REF], or why in general children acquire these sounds later in comparison to stops. For postalveolars, a more frontal articulation for the realisation of voicing versus an articulation further to the back for voiceless can be introduced in speech therapy.

Figure captions Figure 1 :

 captions1 Figure captions

Figure 2 :

 2 Figure 2: Bar plots showing averaged ANT in % (grey) and COG*9 (black), error bars

Figure 3 :

 3 Figure 3: Example for frequency of contact plot for DP's realisation of initial /z/ (left) versus

Figure 4 :

 4 Figure 4: Bar plots showing averaged ANT in % (grey) and COG*9 (black), error bars

  Figure 1

Table 2

 2 Correlation coefficients after Spearman-Rho, significant correlations are printed in bold.

	Correlation		/s/ & /z/			/S/ & /Z/
	Coefficients	ANT & voicdur_rel	PC & voicdur_rel
		COG & voicdur_rel	COG & voicdur_rel
	Speaker	n	R	P-value	n	R	P-value
	BM	140	-0.149	0.078	49	-0.120	0.413
			-0.113	0.183		0.203	0.162
	CF	140	-0.119	0.162	50	-0.052	0.721
			-0.119	0.160		0.081	0.577
	DM	137	0.324	>0.001	50	0.225	0.115
			0.484	>0.001		0.526	>0.001
	JF	139	0.415	>0.001	50	0.446	0.001
			0.341	>0.001		0.357	0.011
	JM	139	0.655	>0.001	50	0.474	0.001
			0.567	>0.001		0.566	>0.001
	RM	140	0.398	>0.001	50	0.415	0.003
			0.589	>0.001		0.365	0.009
	SF	140	0.195	0.021	50	0.355	0.011
			0.013	0.878		0.435	0.002

Acknowledgements

This work was supported by a grant from the German Research Council (DFG) GWZ 4/8-1, P.1. We would like to thank our subjects, Jörg Dreyer for technical expertise, the editors of this special issue, two anonymous reviewers, Christine Mooshammer, Pascal Perrier, Pauline Welby, Phil Hoole, Bernd Pompino-Marschall, Martine Toda for comments on an earlier version of this paper, and John Tammena for proof-reading. This work is dedicated to Dieter Fuchs.

Footnotes

1. Based on a questionnaire (2004), 84% of the 4564 German participants pronounced the word cent with initial [s], 9.8% with [ts], and 6.2% used both forms (see http://www.sprechwiss.uni-halle.de/aktuelles/index.html). Since in the majority of cases the fricative instead of the affricate was chosen, we included the word in our corpus.

Appendix


Véronique Zamboni

Sarah Barelier

Robert Dixon

Nicola Galley

Amani Ghanem

Phong Nguyen

Héloize Cahuzac

Bartłomiej Salamaga

Peter J Davis

Stéphane Mesnage
email: s.mesnage@sheffield.ac.uk

Florence Vincent

Molecular basis for substrate recognition and septum cleavage by AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis

Keywords: glycosyl hydrolase, structure-function, crystal structure, enterococcus, cell wall, peptidoglycan

The cleavage of septal peptidoglycan at the end of cell division leads to the separation of daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death. Their activity must therefore be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains that can no longer disseminate in the host and are readily taken up by phagocytes. AtlA has structural homologs in other important pathogens such as Listeria monocytogenes and Salmonella typhimurium and therefore represents an attractive model to design new inhibitors of pathogenesis. Here, we provide a 1.6Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved β-hairpin and a complex network of hydrogen bonds that bring two catalytic residues at an ideal distance for an inverting mechanism. Based on the model of the AtlA-substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth.

Introduction

Enterococcus faecalis is a nosocomial pathogen causing a wide range of infections, some of which can be life-threatening [START_REF] Arias | The rise of the Enterococcus: beyond vancomycin resistance[END_REF]. The virulence of this organism relies on a combination of several factors acting synergistically to outcompete other organisms and cause the disease. These factors include the capacity to survive abiotic stresses (such as pH changes or exposure to bile salts) and antibiotic treatments (e.g., cephalosporins) [START_REF] Arias | The rise of the Enterococcus: beyond vancomycin resistance[END_REF][START_REF] Bradley | Heat and chemical resistance of enterococci[END_REF]. During pathogenesis, E. faecalis has evolved several mechanisms that enable this organism to evade the host innate immune system. These include the production of two distinct cell surface polymers (a capsule and a rhamnopolysaccharide) that play an antiphagocytic role (for review, see [START_REF] Ramos | Sugarcoating it: Enterococcal polysaccharides as key modulators of host-pathogen interactions[END_REF]). Another mechanism relies on the control of cell chain length. E. faecalis has a distinctive morphology and forms diplococci and short cell chains made of 4 to eight cells. The disruption of cell separation at the end of the division, leading to the formation of cell chains, has a dramatic impact on virulence. It prevents the dissemination of bacteria from causing a systemic infection and leads to the uptake of cell chains by phagocytes [START_REF] Salamaga | Bacterial size matters: Multiple mechanisms controlling septum cleavage and diplococcus formation are critical for the virulence of the opportunistic pathogen Enterococcus faecalis[END_REF].

One peptidoglycan hydrolase, AtlA, has a predominant role in E. faecalis septum cleavage. atlA mutants are viable but form extremely long chains and are greatly attenuated in the zebrafish virulence model of infection [START_REF] Salamaga | Bacterial size matters: Multiple mechanisms controlling septum cleavage and diplococcus formation are critical for the virulence of the opportunistic pathogen Enterococcus faecalis[END_REF]. It has also been shown that AtlA activity contributes to the bactericidal activity of beta-lactam antibiotics [START_REF] Bravetti | Contribution of the autolysin AtlA to the bactericidal activity of amoxicillin against Enterococcus faecalis JH2-2[END_REF][START_REF] Dubee | The in vitro contribution of autolysins to bacterial killing elicited by amoxicillin increases with inoculum size in Enterococcus faecalis[END_REF]. Based on the contribution of AtlA to pathogenesis and antibiotics-mediated killing, this peptidoglycan hydrolase represents an attractive target to prevent enterococcal infections whilst limiting microbiota changes.

AtlA is a multimodular N-acetylglucosaminidase belonging to the glycosyl-hydrolases GH73 family in the CAZY database [START_REF] Drula | The carbohydrate-active enzyme database: functions and literature[END_REF]. To avoid AtlA-mediated autolysis, the activity of AtlA is tightly controlled, both spatially and temporally [START_REF] Salamaga | Bacterial size matters: Multiple mechanisms controlling septum cleavage and diplococcus formation are critical for the virulence of the opportunistic pathogen Enterococcus faecalis[END_REF]. The enzyme undergoes post-translational modifications (glycosylation and proteolytic cleavage) and preferentially binds to denuded glycan chains (preferentially found at the mid-cell) via the C-terminal LysM domain [START_REF] Eckert | Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis[END_REF].

In this study, we report the first structure that captures a closed conformation of the active site, revealing a single-displacement (inverting) mechanism. We further provide a structure-function analysis of AtlA catalytic domain. We demonstrate the endo-glucosaminidase activity and identify the critical residues for catalysis both in vitro and in the context of the live bacteria.

Based on these data, we propose a model for the interaction of AtlA with peptidoglycan, a first step towards the rational design of specific inhibitors targeting an enterococcal virulence factor.

Results

Overall Structure of AtlAc

The structure of AtlAc (161 residues) was solved at a resolution of 1.45Å. It adopts a lysozyme This fold superimposes with Auto from Listeria monocytogenes with a rmsd of 1.02Å for 107 Cα atoms and with FlgJ from S. thyphimurium (FlgJ St ) with a rmsd of 2.2Å for 119Cα , which account for the larger opening of the β-hairpin (Figure 1B) [START_REF] Bublitz | Structural basis for autoinhibition and activation of Auto, a virulence-associated peptidoglycan hydrolase of Listeria monocytogenes[END_REF]. The structure also contains 5 glycerol molecules coming from the cryogenic conditions. Only one polypeptide of AtlAc is found in the asymmetric unit, suggesting that the protein is monomeric in solution.

The AtlAc active site shows two highly conserved glutamate residues: Glu212 is equivalent to L. monocytogenes Auto Glu122 and FlgJ St E184 catalytic residues and is located at a similar position, at the end of helix α1 (Figure 1B). The second predicted catalytic glutamate, Glu246, stands on β2 and is equivalent to Glu156 in L. monocytogenes Auto and Glu223 in FlgJ St . Unlike Auto and FlgJ St which show an open groove for the active site, the AtlAc active site shapes into a tunnel (Figure 1B and1D). The β-hairpin closing on the α-lobe by forming several hydrogen bonds: Glu246 OE2 on strand β1 with Lys232 NZ on a loop between α2 and β1, Glu246 with Thr312 OG1 and the water molecule 12 (Wat12), and Thr312 OG1 with Tyr247 main chain N (Figure 1C). Lys232 makes also a bond with OE1 Glu257 on β2, but this Glu residue is not conserved in the GH73 family (Figure 1C and2).

Several aromatic residues that have been identified in the GH73 family to be essential for substrate binding or catalysis, are also conserved in AtlA active site, namely Tyr310 which belong to the YATD motif, a signature motif of the GH73 family, Tyr316, Phe259 and Phe229 (Figure 1C and 2) [START_REF] Lipski | Structural and biochemical characterization of the beta-Nacetylglucosaminidase from Thermotoga maritima: toward rationalization of mechanistic knowledge in the GH73 family[END_REF].

PG hydrolysis by AtlA

The crystal structure of AtlAc revealed a partial occlusion of the catalytic tunnel by the Lys232 residue (Figure 1C). Based on the analysis of other GH73 catalytic domains, we hypothesized that this residue is flexible to allow the recognition of long glycan chains. An alternative explanation could be that AtlA displays exo-glucosaminidase activity, thereby accommodating a smaller substrate in the catalytic tunnel. To test our hypothesis, we investigated whether AtlA displays exo-or endo-glucosaminidase activity. Purified peptidoglycan sacculi were incubated in the presence of increasing amounts of recombinant AtlA and solubilized muropeptides were analysed by rp-HPLC. We anticipated that an exo-glucosaminidase would release monomeric peptidoglycan fragments, whilst an endo-glucosaminidase activity would release monomeric and multimeric structures simultaneously. The comparison of muropeptide profiles suggested that AltlA displays endo-glucosaminidase activity as it generated the same proportion of muropeptides with different oligomerization states, irrespective of the amount of enzyme added (Figure 3).

Docking of a peptidoglycan substrate to the active site of AtlA

To gain insight into AtlA catalytic mechanism and peptidoglycan binding mode, a peptidoglycan fragment was docked to the crystal structure of the enzyme. We used a tetrasaccharide chain (MurNAc -2 -GlcNAc -1 -MurNAc +1 -GlcNAc +2 ) with MurNAc residues substituted by short tripeptide stems found in E. faecalis peptidoglycan (L-Ala-γD-Gln-L-Lys) and amenable to docking experiments (Figure S1). The ligand was docked to a grid that encompasses the putative active site of AtlA and includes the two predicted catalytic glutamates, Glu212 and Glu246. The protein was kept rigid except for Lys232, which blocks part of the active site tunnel and has to be flexible to accommodate the substrate and allow AtlA endo-glucosaminidase activity. The docking produced 8 poses for the ligand with binding energies ranging from -6.5 to -5.4 kcal/mol. Figure 4A shows the docked complex with the ligand in its best (lowest energy) binding pose. Interestingly, docking experiments using peptidoglycan ligands with shorter side chains (L-Ala-D-Gln, or L-Ala) produced the same pose as for the longer side chain (Figure S2).

The ligand adopts an extended conformation that spans the active site of AtlA, going through the tunnel that contains the two putative catalytic glutamates. The oxygen of Glu212 is located 2.8 Å away from the glycosidic bond, in agreement with the cleavage of the glycosidic bond between GlcNAc -1 and MurNAc +1 . The ligand mainly interacts via the GlcNAc -1 moiety, making several hydrogen bonds with the backbones of Gly230, Trp245, Tyr310, Thr312, Asp313 and with the side chain of Glu246 (Figure 4B), suggesting that the MurNAc +1 -GlcNAc +2 part of the substrate is the first to leave the active site after cleavage. The other sugar moieties of the ligand also establish hydrogen bonds with the protein via MurNAc -2 (with Tyr310), MurNAc +1 (with Glu212) and GlcNAc +2 (with Asp243). Although potential interactions may involve the amino acid side chains of the ligands (with residue Val211, for example), the flexibility of the peptide stems renders such predictions unreliable.

Identification of AtlA residues important for activity in vitro

Based on the structural analysis of the AtlA catalytic domain and the docking of a peptidoglycan fragment, we investigated the role of several residues potentially involved in substrate recognition and catalysis. These included the catalytic glutamate residue Glu246 that had not been previously studied, Lys232, Phe259, Thr312 and Tyr316. Tyr247 was added as a control, as this residue is not expected to contribute to the catalytic activity of AtlA. The expression plasmid encoding full length recombinant AtlA was mutagenized to introduce these 5 mutations in the catalytic domain. All recombinant proteins were expressed as soluble proteins and were purified by affinity chromatography (Fig. 5A). Circular dichroism confirmed that the mutations had no major impact on the overall fold of the recombinant proteins (Fig. S3). Using M. lysodeikticus autoclaved cells as a substrate, we determined the specific activity of individual mutants using the spectrophotometric assay previously described (Fig. S4). The Tyr247Ser and Tyr316Ser mutations had a limited impact on in vitro specific activity (2.2-fold and 2.4-fold decrease, respectively) whilst the four other mutations (Lys232Ala, Phe259Val, Thr312Val and Glu246Gln) had a pronounced impact, leading to residual activities between 2.5% to 5.3% when compared to the WT enzyme (Fig. 5B).

Figure 5. Purification and characterisation of recombinant AtlA enzymatic activity. A,

SDS-PAGE analysis of full length recombinant AtlA proteins. AtlA variants were produced in E. coli and purified using a one-step immobilized metal affinity chromatography; lane 1, WT; lane 2, K232A; lane 3, Y247S; lane 4, F259V; lane 5, T312V; lane 6, Y316S; lane 7, E246Q. B, specific activity (expressed as Δo600nm/h/mg) was measured using M. luteus as a substrate.

Identification of AtlA residues important for septum cleavage

AtlA is a secreted autolysin that undergoes posttranslational modifications including proteolytic cleavage and glycosylation (4). To confirm the results obtained with recombinant proteins in vitro, we sought to measure the impact of mutations using cell chain length as a readout of enzymatic activity (Fig. 6). Each mutation was introduced on the chromosome by allelic exchange and the forward scattered light associated with mutant cells was measured by flow cytometry to investigate the capacity of individual AtlA mutant proteins to cleave the septum. All mutant proteins were produced at a similar level, indicating that mutations had very little (if any) impact on stability (Fig. 6A). The results were consistent with previous enzymatic assays and the docking model. Tyr247Ser has no impact on septum cleavage, confirming that the mutation does not affect the structure of the enzyme. The other five mutations had a clear impact on septum cleavage, forming cell chains significantly longer than the wild type (Fig. 6B). As expected, Glu212Gln and Glu246Gln mutations had the most pronounced impact on AtlA activity, leading to the formation of extremely long cell chains. Both Phe259 and Lys232 were shown to be critical for AtlA activity, whilst Thr312 had a relatively smaller role.

Discussion

The enzymatic assays using recombinant AtlA and measurement of septum cleavage in strains expressing AtlA variant provided insightful information to propose a catalytic mechanism.

Glu212 is strictly conserved in the GH73 enzyme family and was previously identified as an essential catalytic residue (4). It is positioned at the end of helix α2, on a highly conserved α-lobe and superimposes with Glu122 from L. monocytogenes Auto (9), and Glu184 from FlgJ St [START_REF] Zaloba | Structural and Biochemical insights into the peptidoglycan hydrolase domain of FlgJ from Salmonella typhimurium[END_REF] (Figure 1B). AtlA Glu212 can therefore be confidently identified as the catalytic proton donor. A second glutamate, Glu246, is positioned on strand β1 like Glu156 in Auto and Glu223 in FlgJ St (Figure 1B et D) and has been proposed to be a nucleophile/base catalyst. This second glutamate residue is conserved only in a subgroup of proteobacteria and the bacteroidetes as mentioned in the phylogenetic study that classified the GH73 sequences into 5 different clusters [START_REF] Lipski | Structural and biochemical characterization of the beta-Nacetylglucosaminidase from Thermotoga maritima: toward rationalization of mechanistic knowledge in the GH73 family[END_REF]. In clusters 1 and 2, GH73 enzymes possess a β-lobe that folds into an extended β-hairpin that contributes to shaping the active site and harbours a conserved glutamate [START_REF] Lipski | Structural and biochemical characterization of the beta-Nacetylglucosaminidase from Thermotoga maritima: toward rationalization of mechanistic knowledge in the GH73 family[END_REF]. This β-hairpin has been modelled in Auto, FlgJ St , and more recently in Sphingomonas sp. FlgJ but is often incomplete in other structural homologs like TM0633, due to its flexibility [START_REF] Bublitz | Structural basis for autoinhibition and activation of Auto, a virulence-associated peptidoglycan hydrolase of Listeria monocytogenes[END_REF][START_REF] Lipski | Structural and biochemical characterization of the beta-Nacetylglucosaminidase from Thermotoga maritima: toward rationalization of mechanistic knowledge in the GH73 family[END_REF][START_REF] Zaloba | Structural and Biochemical insights into the peptidoglycan hydrolase domain of FlgJ from Salmonella typhimurium[END_REF][START_REF] Hashimoto | Crystal structure of the glycosidase family 73 peptidoglycan hydrolase FlgJ[END_REF]. In Auto and FlgJ St structures, the position of the β-hairpin places the two catalytic glutamates at 13Å and 20Å apart respectively, and this is due to the presence of an α-helix or a β-strand of a neighbouring symmetry monomer that occludes their active sites [START_REF] Bublitz | Structural basis for autoinhibition and activation of Auto, a virulence-associated peptidoglycan hydrolase of Listeria monocytogenes[END_REF][START_REF] Zaloba | Structural and Biochemical insights into the peptidoglycan hydrolase domain of FlgJ from Salmonella typhimurium[END_REF][START_REF] Hashimoto | Crystal structure of the glycosidase family 73 peptidoglycan hydrolase FlgJ[END_REF] 7A). In AtlA and Auto, Lys232 and Lys142 make electrostatic bonds with the nucleophiles Glu246 and Glu156 respectively (yellow dashes lines). In FlgJ St , Lys207 is too far to bind to Glu223. B, AtlA is an Nacetylglucosaminidase hydrolysing the β-1,4 linkage between NAG and MurNAc using an inverting mechanism. Glu246 (catalytic base) activate a wat12 by abstracting a proton, which enable the water molecule to make a nucleophilic attack on the anomeric carbon and lead to the formation of an oxocarbenium ion-like transition state. Glu212 play the role of an acid that will donate a hydrogen to the scissile glycosidic bond. This glycolysis produces an α-anomeric configuration of the NAG at the reducing end.

(Fig 1B)(Figure
In these structures, the β-hairpin is widely open, and the authors suggest that the β-hairpin takes this position to accommodate the substrate in the active site. In AtlAc, the β-hairpin shows a "closed" conformation and the distance between Glu212 and Glu246 is 9 Å, which is the distance required for a classic type 2 nucleophile substitution with an inverting mechanism (14) (Figure 7A). We, therefore, propose that Glu212 acts as a general acid to protonate the glycosidic oxygen, favouring the aglycon departure, while the general base Glu246 activates a water molecule to make a nucleophile attack on the sugar anomeric carbon. Glu246 OE1 is making a hydrogen bond with a water molecule (Wat12) and Lys232 (Figure 7B). This water molecule could therefore be activated by Glu246 if the latter encounters a displacement of a negative charge and takes a proton from Wat12, which will, in turn, be able to perform the nucleophilic attack on the anomeric carbon of the substrate. In previous studies where structural homologs show a β-hairpin widely open, the authors proposed that the β-hairpin will undergo several conformational changes to accommodate the substrate in the active site and subsequently place the two catalytic glutamate residues at the proper distance for hydrolysis of the glycosidic bond [START_REF] Mccarter | Mechanisms of enzymatic glycoside hydrolysis[END_REF]. Our crystallographic data strongly support this model and suggest a contribution of a conserved lysine residue (Lys232) in this process, as demonstrated by the loss of AtlA activity associated with the Lys232 mutation both in vitro and in vivo. The structure of Auto illustrates an intermediary position of the β -hairpin where the Lys142 can reach the catalytic Glu and make electrostatic contact to initiate the closing of the β -hairpin. Our AtlAc crystal structure shows the electrostatic bond between Lys232 and Glu246 maintaining the position of the β-hairpin resulting in a closed active site (Figure 7A). We propose that this electrostatic contact between the Lys232 and the Glu246 stabilizes the charges to help its attack on the water molecule. The identification of the endoglucosaminidase activity together with modelling of the tetrasaccharide peptide bound in the active site indicates that after the movement of the β-hairpin, Lys232 adopts another conformation to allow substrate binding and catalysis using an inverting mechanism.

Collectively, our results indicate that AtlA uses an inverting mechanism to hydrolyse the β-1,4 glycosidic bond.

Experimental procedures

Bacterial strains, plasmids, and growth conditions. All strains and plasmids used in this study are described in Table S1. The bacteria were grown at 37°C in Brain Heart Infusion broth or agar (15 g/l) (BHI, Difco laboratories, Detroit, USA). When required, E. coli was grown in the presence of 100 µg/ml ampicillin (for protein expression) or 200 µg/ml erythromycin (for pGhost selection). E. faecalis transformants were selected with 30 µg/ml erythromycin.

Plasmid construction. pML420 was built to express AtlA catalytic domain. The DNA fragment encoding amino acids 172 to 337 was PCR amplified from V583 genomic DNA [START_REF] Paulsen | Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis[END_REF] with oligonucleotides EF Atla_cat_F and Atla_cat_R (Table S1) using Vent DNA polymerase (NEB).

The resulting fragment was cloned in frame with the hexahistidine sequence of pET2818, a pET2816b derivative (8), using NcoI and BamHI. Plasmids expressing full-length AtlA derivatives (with single amino-acid substitutions) were constructed by site-directed mutagenesis using plasmid pML118 as a template [START_REF] Eckert | Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis[END_REF] following the quickchange mutagenesis protocol [START_REF] Liu | An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol[END_REF] and oligonucleotides described in Table S1. pML118 derivatives were used as a template to amplify the DNA fragments encoding atlA alleles. The corresponding PCR products were cloned in pGhost9 using XhoI and EcoRI.

Construction of E. faecalis mutants by allele exchange. The protocol described previously was followed [START_REF] Mesnage | Role of Nacetylglucosaminidase and N-acetylmuramidase activities in Enterococcus faecalis peptidoglycan metabolism[END_REF]. E. faecalis was electroporated with pGhost9 derivatives and transformants were selected at 30°C in the presence of erythromycin. Single crossing-overs were induced at nonpermissive temperature (42°C) and screened by PCR. The second recombination event was triggered by subculturing recombinant clones in BHI at 42°C. Erythromycin sensitive colonies were screened by PCR to identify mutants.

Production and purification of recombinant proteins.

E. coli Rosetta(DE3) pLysS cells (Novagen) containing recombinant pET vectors were cultured in ZY auto-induction medium (18) containing 100 mg/ml of ampicillin and 34 mg/ml of chloramphenicol at 37 °C until the OD600 reaches 0.6. The cultures were then incubated overnight at 17 °C. Cells were harvested by centrifugation and the frozen pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 8, 300 mM NaCl, 10 mM imidazole, 1mM EDTA, 0.25 mg/ml Lysozyme, 5% glycerol, 0.1% triton). After a 30 min incubation with 0.1 mg/ml deoxyribonuclease and 0.02M magnesium sulphate at 4°C, the cells were disrupted by sonication on ice. The lysate was clarified by centrifugation and the supernatant was applied onto a nickel-chelate affinity resin using an ÄktaXpress (Cytiva). The resin was washed with 50 mM Tris-HCl pH 8, 300 mM NaCl, and 50 mM imidazole. The protein was eluted with 50 mM Tris-HCl pH 8, 300 mM NaCl, and 250 mM imidazole and dialyzed against the gel filtration buffer (20 mM Mes pH 6, 100 mM NaCl and 1mM DTT). The protein was further purified on a Superdex 75 16/60 column (Cytiva). Pure fractions, as analyzed by SDS-PAGE, were pooled.

Circular Dichroism Spectroscopy

CD samples were diluted to a concentration of 0.25mg/ml in 25 mM Tris-HCL + 150mM NaCl buffer, pH 8.0. Far-UV CD spectra were acquired with a Jasco J-810 spectropolarimeter using a 0.1 cm pathlength cell. Spectra were acquired with a Peltier thermally controlled cuvette holder at 25°C, from 190 to 350 nm, with a data pitch of 1 nm at a rate of 50 nm/min, a response time of 8 seconds and averaged over 5 accumulations. Mean residue ellipticity, [Θ] MRW (deg cm 2 dmol -1 ) was calculated to allow reliable comparison of different proteins using the equation:

where Θ is the measured ellipticity in degrees, M r is the protein molecular weight in kDa, c is the protein concentration in mg/ml, d is the pathlength in centimetres, N A is the number of amino acids per protein. The factor of 100 converts molar concentration to dmol/cm 3 [START_REF] Creighton | Protein structure: a practical approach[END_REF].

Crystallization, data collection and processing

The catalytic domain of AtlA (AtlAc) was concentrated to 14 mg/ml using a 10 kDa cut-off ultracentrifugation membrane (Thermoscientific). Crystals were obtained at 20°C by screening

The Wizard Classic crystallization kit (Molecular Dimensions TM ) using a nanoliter sitting drops setup with automated crystallization TECAN Genesis and ttplabtech Mosquito robots. A total of 300 μl of AtlAc (14 mg ml/1) was mixed to 100 μl of reservoir solution composed of 0.2 M Magnesium chloride, 10% polyethylene glycol (PEG) 3000 and 0.1M Na-Cacodylate pH 6.5. A single crystal of AtlAc was mounted on a rayon loop, soaked in a cryoprotectant solution composed of the crystallization conditions with 23% glycerol and subsequently flash-cooled to 120 K. A data set was collected at 1.4 Å at the ID30B beamline (ESRF, France). Data were integrated, scaled and reduced with XDS (20) and AIMLESS [START_REF] Dodson | Collaborative Computational Project, number 4: providing programs for protein crystallography[END_REF]. Unless otherwise cited, all further crystallographic computations were carried out using the CCP4 suite of programmes [START_REF] Dodson | Collaborative Computational Project, number 4: providing programs for protein crystallography[END_REF].

Phasing, Model Building, and Refinement

The structure was solved by molecular replacement, using MolRep [START_REF] Vagin | Molecular replacement with MOLREP[END_REF], with a search model of AtlAc built by Modeller using Auto structure from L. monocytogenes (54% sequence identity with AtlAc) as a template (PDB 3fi7) [START_REF] Bublitz | Structural basis for autoinhibition and activation of Auto, a virulence-associated peptidoglycan hydrolase of Listeria monocytogenes[END_REF][START_REF] Webb | Comparative protein structure modeling using Modeller[END_REF]. The solution of MolRep gave a correlation coefficient of 0.47 for one monomer in the asymmetric unit. The electron density map calculated from the model was of sufficient quality to allow tracing of 99% of the molecule (156 residues) using the REFMAC/ARP-wARP programs [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF][START_REF] Perrakis | ARP/wARP and molecular replacement[END_REF]. Unless otherwise cited, all further crystallographic computations were carried out using the CCP4 suite of programs [START_REF] Dodson | Collaborative Computational Project, number 4: providing programs for protein crystallography[END_REF]. The model was refined using REFMAC [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF] with manual correction using Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF]. The final model contains 1281 non-hydrogen protein atoms with 182 water molecules and 5 glycerol molecules.

The crystallographic Rcryst and Rfree values are 14 and 17%, respectively (see Table 1).

Docking experiments

Molecular docking was performed in Autodock Vina [START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[END_REF]. The structure of Atla was used as a docking model. Ligand and receptor structures were prepared in Jligand [START_REF] Lebedev | JLigand: a graphical tool for the CCP4 template-restraint library[END_REF] and AutoDockTools [START_REF] Morris | AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility[END_REF]. Partial charges were calculated using Gasteiger-Hückel method. Exhaustiveness was set to 8 and default parameters were used unless otherwise stated. The ligand was treated as flexible, and the protein was kept rigid during the docking runs, except for the side chain of Lys232, which was treated as flexible. The grid size was set to 22 x 30 x 24 (Å) and the grid box's center points were set to target the active site of the protein, with the center at X = 32, Y = 20, Z = -11.5. All 3D and 2D representations of protein-ligand complexes were visualized using PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC) and Ligplot+ [START_REF] Laskowski | LigPlot+: multiple ligand-protein interaction diagrams for drug discovery[END_REF], respectively.

Determination of peptidoglycan hydrolase activity.

Hydrolysis of purified cell walls was measured using an Ultrospec 2000 spectrophotometer (Amersham biosciences, Uppsala, Sweden) and following the decrease in turbidity at 450 nm for 15 min at 37°C in 25 mM Tris-HCl, pH 7.5, 100 mM NaCl buffer. Peptidoglycan was resuspended at a concentration giving an absorbance at 450nm of 0.6. Various dilutions of AtlA and its derivatives were tested to identify conditions in which the velocity of hydrolysis was proportional to enzyme concentration. Enzymatic activity was expressed as A 450 units per min per mmol of protein; the residual activity of individual mutants was expressed as the percentage of wild-type activity.

For zymogram analysis, crude extracts were separated by SDS-PAGE using gels containing 0.2 % autoclaved Micrococcus lysodeikticus cells. After electrophoresis, the proteins were renatured by incubating the gel for 24 h in 25 mM Tris (pH 8.0) buffer containing 0.1 %Triton at 37°C. Lytic activities could be visualized as clear bands on the opaque SDS-PAGE.

Western blot analyses

Cells were grown in BHI to exponential phase. The equivalent of 250µl of acid-washed <106µm glass beads (SIGMA) was added to a 1.5ml aliquot of the culture and cells were broken using a Fastprep homogenizer (MP Biomedicals) with 6 cycles at maximum speed with 1 min pause between cycles. The equivalent of 25µl of culture was loaded on a 12% SDS-PAGE and transferred to nitrocellulose. AtlA was detected using a rabbit polyclonal antibody against the full-length protein as previously described (4).

Flow cytometry and statistical analyses

Overnight static cultures at 37°C were diluted 1:100 into fresh broth (OD600 ~0.02) and grown to mid-exponential phase (OD600~0.2 to 0.4). Bacteria were diluted 1:100 in filtered phosphate buffer saline and analyzed by flow cytometry using a Millipore Guava easyCyte H2L system. Light scatter data were obtained with logarithmic amplifiers for 20,000 events. Forward scattered light values were compared using Graphpad and an unpaired t test with Welch's correction.

  α/β fold with an α-lobe containing 5 α-helices (α1: 180-197, α2: 202-213; α3: 265-278, α4: 299-306, α5:315-327), 5 helices 3 10 , a small β-hairpin (280-286) and a major β-lobe covering the binding site, made of 2 antiparallel β-strands (β1:239-248, β2: 251-260) connecting α2 and α3(Figure1A).

Figure 1 :

 1 Figure 1: AtlAc structure and overlay of AtlAc, Auto and FlgJ St . A, Cartoon representation of the structure of AtlAc. The secondary elements are numbered and the catalytic Glu246 and Glu212 are shown in stick. B, Overlay of AtlAc in white, Auto from L. monocytogene (blue, PDB: 3fi7), and FlgJ from S. thyphimurium (FlgJ St , Green, PDB 5dn5). The conserved putative

Figure 2 :

 2 Figure 2: Structure based sequence alignment of several GH73 enzymes. This alignment has been done with the program Expresso (11). AtlAc secondary structures are drawn at the top of the alignment. The conserved putative catalytic glutamate residues are shown with green dots. The mutated residues used in this study are shown with orange triangles.

Figure 3 .

 3 Figure 3. rp-HPLC analysis of muropeptides solubilised by AtlA. A, E. faecalis peptidoglycan was digested by increasing amounts of recombinant AtlA (from 1µg to 64µg). Soluble fragments released were reduced and separated on a Hypersil C18 column, showing a mixture of monomers and multimers. The schematic structure of major monomers (peaks 1 and 2), dimers (peaks 3 and 4) and trimers (peaks 5 and 6) is shown. GlcNAc and MurNAc are shown as filled squares, peptide stem residues as filled circles. B, All traces were normalised to display the same amplitude for the major dimer, showing no preferential release of monomeric structures and an overall very similar profile, irrespective of the amount of enzyme used.

Figure 4 .

 4 Figure 4. Model of the interaction between a peptidoglycan substrate and Atla. A, Top scoring pose of a peptidoglycan substrate docked to the active site of Atla. The ligand substrate is represented as orange sticks, the receptor is represented as light grey cartoon, the main residues of the active site are shown in sticks and the residues selected for mutational studies are highlighted in blue. B, Ligplot+ representation of the interactions of a peptidoglycan substrate (ligand) with the active site residues of Atla (receptor).

Figure 6 .

 6 Figure 6. Flow cytometry analysis of bacterial cell chain length in E. faecalis strains expressing AtlA mutant alleles. A, Median forward scattered (FSC) light of exponentially growing cells was measured by flow cytometry.The results shown correspond to the average value of six biological replicates for the parental strain (WT), the atlA in-frame deletion mutant (ΔatlA) and the six strains producing AtlA mutant alleles. All cell chain lengths were significantly different from the wild-type strain (****P<0.0001). The amount of protein produced by each strain was checked by Western blot using an anti-AtlA polyclonal serum. B, Median FSC values were compared to that from the ΔatlA mutant; NS, not significant; N/A, not applicable.

Figure 7 :

 7 Figure 7: AtlA catalytic mechanism. A, The motion performed by the β-hairpin to close on the active site is shown by comparing FlgJ St , Auto and AtlA β-hairpin's positions.In AtlA and Auto, Lys232 and Lys142 make electrostatic bonds with the nucleophiles Glu246 and Glu156 respectively (yellow dashes lines). In FlgJ St , Lys207 is too far to bind to Glu223. B, AtlA is an Nacetylglucosaminidase hydrolysing the β-1,4 linkage between NAG and MurNAc using an inverting mechanism. Glu246 (catalytic base) activate a wat12 by abstracting a proton, which enable the water molecule to make a nucleophilic attack on the anomeric carbon and lead to the formation of an oxocarbenium ion-like transition state. Glu212 play the role of an acid that will donate a hydrogen to the scissile glycosidic bond. This glycolysis produces an α-anomeric configuration of the NAG at the reducing end.

Table 1 Structural data collection and refinement statistics Crystal parameters

 1 R cryst =S hkl | |Fo|-|Fc| |/S hkl |Fo| c R free is calculated for randomly selected reflections excluded from refinement

	Space group	P3 1 21
	Cell parameters (Å)	a=b=62.55, c=93.22
	Data quality
	Wavelength (Å)	0.9762 (PX1)
	Resolution of data (Å)	46.8-1.45
	Unique reflections	38042
	R merge (outer shell)	0.06 (0.93)
	Mean I/I (outer shell)	9.8 (1.1)
	Completeness (outer shell) %	99.9 (99.9)
	CC1/2 (outer shell)	0.99 (0.76)
	Multiplicity (outer shell)	4.7 (4.2)
	Refinement
	R cryst b	0.14
	R free c	0.17
	Root mean square deviation 1-2 bonds (Å)	0.012
	Root mean square deviation 1-3 angles (°)	1.539

a R merge = (S hkl S i ½I hkl -(I hkl )½/ S hkl S i [I hkl ]). b

Acknowledgments

This work was supported by an International Exchange grant from the Royal Society to SM and FV (IEC\R2\170260).

Data availability

All data presented in this article are to be shared upon request to Dr Florence Vincent (fvincent.cnrs@univ-amu.fr) or Dr Stéphane Mesnage (s.mesnage@sheffield.ac.uk).

The atomic coordinates of AtlAc were deposited in the PDB (entry 7QFU)

Supporting information

This article contains supporting information (Figures S1, S2, S3, S4 and Table S1).

Author contributions

Conceptualization: SM and FV; Data curation: SB, SM and FV; Formal Analysis: SB, NG, SM and FV; Funding acquisition: SM and FV; Investigation: VZ, SB, RD, NG, AG, PN, CG, BS, PJD, SM and FV ; Methodology: SB and FV; Project administration: SM and FV; Supervision: SM and FV ; Validation : VZ, SB, NG, BS, SM and FV ; Visualization: SB, SM and FV ; Writing -original draft: SM and FV ; Writing -review & editing : SB, SM and FV.