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# The secretome of Thermococcus barophilus in the presence of carbohydrates and the potential role of the TrmBL4 regulator

Maria Batour, Sebastien Laurent, Yann Moalic, Hala Chamieh, Samir Taha, Mohamed Jebbar

<figure><img src="image_2.png" /></figure>

## To cite this version:

Maria Batour, Sebastien Laurent, Yann Moalic, Hala Chamieh, Samir Taha, et al.. The secretome of Thermococcus barophilus in the presence of carbohydrates and the potential role of the TrmBL4 regulator. Environmental Microbiology Reports, 2023, 15 (6), pp.530-544. 10.1111/1758-2229.13186 . hal-04204135

HAL Id: hal-04204135

https://hal.science/hal-04204135

Submitted on 15 Apr 2024

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# The secretome of Thermococcus barophilus in the presence of carbohydrates and the potential role of the TrmBL4 regulator

Maria Batour, Sebastien Laurent, Yann Moalic, Hala Chamieh, Samir Taha, Mohamed Jebbar

► To cite this version:

Maria Batour, Sebastien Laurent, Yann Moalic, Hala Chamieh, Samir Taha, et al.. The secretome of Thermococcus barophilus in the presence of carbohydrates and the potential role of the TrmBL4 regulator. Environmental Microbiology Reports, 2023, 15 (6), pp.530-544. 10.1111/1758-2229.13186 . hal-04204135

HAL Id: hal-04204135

https://hal.science/hal-04204135v1

Submitted on 15 Apr 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH ARTICLE

<figure><img src="image_3.png" /></figure>

ENVIRONMENTAL MICROBIOLOGY
REPORTS

Applied
Microbiology
International

# The secretome of *Thermococcus barophilus* in the presence of carbohydrates and the potential role of the TrmBL4 regulator

Maria Batour<fnref n="1" />,<fnref n="2" /> | Sébastien Laurent<fnref n="1" /> | Yann Moalic<fnref n="1" />,<fnref n="3" /> ID | Hala Chamieh<fnref n="2" /> |
Samir Taha<fnref n="2" /> | Mohamed Jebbar<fnref n="1" /> ID

Correspondence

Mohamed Jebbar, Univ Brest, CNRS, Ifremer,
Laboratoire de Biologie et d'Écologie des
Écosystèmes marins profonds (BEEP), IUEM,
Rue Dumont d'Urville, F-29280 Plouzané,
France.

Email: mohamed.jebbar@univ-brest.fr

Funding information

IsBlue, Grant/Award Number: ANR-17-EURE-0015; *Agence Nationale de la Recherche*, Grant/Award Number: ANR-16-CE12-0016; CNRS, Univ Brest, Ifremer, IsBlue, and the Lebanese University

Abstract

Global transcriptional regulators are crucial for supporting rapid adaptive responses in changing environments. In Thermococcales, the TrmB sugar-sensing regulator family is well represented but knowledge of the functional role/s of each of its members is limited. In this study, we examined the link between TrmBL4 and the degree of protein secretion in different sugar environments in the hyperthermophilic Archaeon *Thermococcus barophilus*. Although the absence of TrmBL4 did not induce any growth defects, proteomics analysis revealed different secretomes depending on the sugar and/or genetic contexts. Notably, 33 secreted proteins present in the supernatant were differentially detected. Some of these proteins are involved in sugar assimilation and transport, such as the protein encoded by *TERMP_01455* (cyclomaltodextrin glucanotransferase), whereas others have intracellular functions, such as the protein encoded by *TERMP_01556* (pyruvate: ferredoxin oxidoreductase Δsubunit). Then, using reverse transcription quantitative polymerase chain reaction experiments, we observed effective transcription regulation by TrmBL4 of the genes encoding at least two ABC-type transporters according to sugar availability.

INTRODUCTION

*Thermococcus barophilus* MP was the first true hyperthermophilic piezophile archaeon isolated from a deep-sea hydrothermal vent (Marteinsson et al., 1999). Previous investigations have shown that its adaptation to high hydrostatic pressure (HHP) is achieved by the modulation of the expression of different gene clusters involved in energy, amino acid, and carbohydrate metabolism (Vannier et al., 2015). Notably, the maltose/maltodextrin ABC transporter (MD system) *TERMP_(01653-01658)* involved in carbohydrate metabolism is one of the HHP-responsive gene clusters (Vannier et al., 2011). The expression of this MD system increases under high pressure, as does the expression of two other carbohydrate ABC

transporters, *TERMP_(00275-00281)* and *TERMP_(01836-01840*; Vannier et al., 2015). Interestingly, the operon *TERMP_(00275-00281)* includes a transcriptional regulator gene (*TERMP_00277*) that encodes TrmBL4 (TrmB-like protein 4), which is a family member of the global transcriptional regulator TrmB, known for its regulation of sugar metabolism. TrmB is especially known to be a repressor and/or activator of sugar ABC transporters and as a histone-like chromosomal stabilizer in Archaea (Gindner et al., 2014; Kim et al., 2016).

Proteins of the TrmB family (TrmB, TrmBL1, TrmBL2, TrmBL3, TrmBL4, and TrmBL5) are present in 13 groups of Euryarchaeota (Lee et al., 2008). *Pyrococcus furiosus* has been shown to possess the genes *trmB* (*PF1743*), *trmBL1* (*PF0124*), *trmBL2* (*PF0496*),

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.

© 2023 The Authors. *Environmental Microbiology Reports* published by Applied Microbiology International and John Wiley & Sons Ltd.

<footnote n="1">¹Univ Brest, CNRS, Ifremer, Laboratoire de Biologie et d'Écologie des Écosystèmes marins profonds (BEEP), IUEM, Plouzané, France</footnote>

<footnote n="2">²Laboratory of Applied Biotechnology, Azm Center for Research in Biotechnology and Its Applications, Lebanese University, Tripoli, Lebanon</footnote>

<footnote n="3">³LabISEN, Yncréa Ouest, Brest, France</footnote>
RESEARCH ARTICLE

<figure><img src="image_2.png" /></figure>

ENVIRONMENTAL MICROBIOLOGY
REPORTS

Applied
Microbiology
International

# The secretome of *Thermococcus barophilus* in the presence of carbohydrates and the potential role of the TrmBL4 regulator

Maria Batour<fnref n="1" />,<fnref n="2" /> | Sébastien Laurent<fnref n="1" /> | Yann Moalic<fnref n="1" />,<fnref n="3" /> ID | Hala Chamieh<fnref n="2" /> |
Samir Taha<fnref n="2" /> | Mohamed Jebbar<fnref n="1" /> ID

Correspondence

Mohamed Jebbar, Univ Brest, CNRS, Ifremer,
Laboratoire de Biologie et d'Écologie des
Écosystèmes marins profonds (BEEP), IUEM,
Rue Dumont d'Urville, F-29280 Plouzané,
France.

Email: mohamed.jebbar@univ-brest.fr

Funding information

IsBlue, Grant/Award Number: ANR-17-EURE-0015; *Agence Nationale de la Recherche*, Grant/Award Number: ANR-16-CE12-0016; CNRS, Univ Brest, Ifremer, IsBlue, and the Lebanese University

Abstract

Global transcriptional regulators are crucial for supporting rapid adaptive responses in changing environments. In Thermococcales, the TrmB sugar-sensing regulator family is well represented but knowledge of the functional role/s of each of its members is limited. In this study, we examined the link between TrmBL4 and the degree of protein secretion in different sugar environments in the hyperthermophilic Archaeon *Thermococcus barophilus*. Although the absence of TrmBL4 did not induce any growth defects, proteomics analysis revealed different secretomes depending on the sugar and/or genetic contexts. Notably, 33 secreted proteins present in the supernatant were differentially detected. Some of these proteins are involved in sugar assimilation and transport, such as the protein encoded by *TERMP_01455* (cyclomaltodextrin glucanotransferase), whereas others have intracellular functions, such as the protein encoded by *TERMP_01556* (pyruvate: ferredoxin oxidoreductase Δsubunit). Then, using reverse transcription quantitative polymerase chain reaction experiments, we observed effective transcription regulation by TrmBL4 of the genes encoding at least two ABC-type transporters according to sugar availability.

INTRODUCTION

*Thermococcus barophilus* MP was the first true hyperthermophilic piezophile archaeon isolated from a deep-sea hydrothermal vent (Marteinsson et al., 1999). Previous investigations have shown that its adaptation to high hydrostatic pressure (HHP) is achieved by the modulation of the expression of different gene clusters involved in energy, amino acid, and carbohydrate metabolism (Vannier et al., 2015). Notably, the maltose/maltodextrin ABC transporter (MD system) *TERMP_(01653-01658)* involved in carbohydrate metabolism is one of the HHP-responsive gene clusters (Vannier et al., 2011). The expression of this MD system increases under high pressure, as does the expression of two other carbohydrate ABC

transporters, *TERMP_(00275-00281)* and *TERMP_(01836-01840*; Vannier et al., 2015). Interestingly, the operon *TERMP_(00275-00281)* includes a transcriptional regulator gene (*TERMP_00277*) that encodes TrmBL4 (TrmB-like protein 4), which is a family member of the global transcriptional regulator TrmB, known for its regulation of sugar metabolism. TrmB is especially known to be a repressor and/or activator of sugar ABC transporters and as a histone-like chromosomal stabilizer in Archaea (Gindner et al., 2014; Kim et al., 2016).

Proteins of the TrmB family (TrmB, TrmBL1, TrmBL2, TrmBL3, TrmBL4, and TrmBL5) are present in 13 groups of Euryarchaeota (Lee et al., 2008). *Pyrococcus furiosus* has been shown to possess the genes *trmB* (*PF1743*), *trmBL1* (*PF0124*), *trmBL2* (*PF0496*),

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.

© 2023 The Authors. *Environmental Microbiology Reports* published by Applied Microbiology International and John Wiley & Sons Ltd.

<footnote n="1">¹Univ Brest, CNRS, Ifremer, Laboratoire de Biologie et d'Écologie des Écosystèmes marins profonds (BEEP), IUEM, Plouzané, France</footnote>

<footnote n="2">²Laboratory of Applied Biotechnology, Azm Center for Research in Biotechnology and Its Applications, Lebanese University, Tripoli, Lebanon</footnote>

<footnote n="3">³LabISEN, Yncréa Ouest, Brest, France</footnote>
<figure><img src="image_4.png" /></figure>

and *trmBL3* (*PF066*; Lee et al., 2008). Transcription assays show that *TrmB* of *P. furiosus* acts as a transcriptional repressor for the trehalose/maltose transport system (TM system) with high DNA binding affinity and for the MD system with lower affinity; this inhibition can be removed by the addition of specific sugars (Lee et al., 2005). Although, the *TrmBL1* of *P. furiosus* acts as a repressor of genes of the MD system and glycolytic enzymes, and as an activator of genes involved in glucogenic pathways, it is inactivated by maltose and maltotriose (Lee, Surma, et al., 2007). Only preliminary experiments have been done on *TrmBL2*, which appears to recognize TM and MD promoters and to play a role at the DNA level, clumping it into dense fibrous structures (Maruyama et al., 2011). The *TrmB* family performs a multiplicity of regulations by acting on the same promoters: at least three regulators, *TrmB*, *TrmBL1*, and *TrmBL2*, regulate gene expression at the level of the MD promoter in *P. furiosus* (Lee et al., 2008). Interestingly, this species can grow on carbohydrates such as cellobiose, maltose, and starch (Kengen et al., 1993; Schmid et al., 2013), which implies the secretion of enzymes such as amylopullulanase (Schmid et al., 2013) and an α-glycosidase (Constantino et al., 1990).

The *trmB* gene in *Thermococcus litoralis* was characterized as a repressor of the TM system (Lee et al., 2003). The role of the other regulators (*TrmBL3*, *TrmBL4*, and *TrmBL5*) in the Thermococcales (Gindner et al., 2014) remains unclear. *Thermococcus barophilus* MP, the first true hyperthermophilic piezophile archaeon isolated from a deep-sea hydrothermal vent (Marteinsson et al., 1999), possesses the *TERMP_01972* gene encoding *TrmB*, which might regulate the adjunct operon *TERMP_(01968-01973*; TM system); *TrmBL2* encoded by the *TERMP_00762* gene; *TrmBL4* encoded by the *TERMP_00277* gene, which can regulate the operon *TERMP_(00278-00281*) in the vicinity and divergently oriented (Figure 1A); *TrmBL1* encoded by the *TERMP_01652* gene, which could regulate the operon *TERMP_(01653-01658*; MD system; Figure 1B); and *TrmBL5* encoded by the *TERMP_01835* gene, which might be involved in the regulation of the *TERMP_(01835-01840*) cluster in *T. barophilus* (Figure 1C; Kim et al., 2016).

The binding sites of *TrmB* and *TrmBL1* were identified in *P. furiosus* (*TrmB*: TACTN₃AGTA for the TM system and TACT for the MD system; *TrmBL1*: TATCACN₅GTGATA for the MD system) as was the binding site of *TrmB* in *T. litoralis* (TACTN₃AGTA for the TM system; Lee et al., 2005). In 2006, Van de Werken et al. identified a conserved palindromic sequence, the Thermococcales glycolytic motif (TGM), TATCACN₅GTGATA, present upstream of all genes involved in sugar metabolism in several Thermococcales species as well as in the promoter region of the

MD operon, but not in the TM operon (Van de Werken et al., 2006). It has been shown that *TrmB* preferentially recognizes the TGM sequence (Lee, Seitz, et al., 2007), while *TrmBL1* target binding is not restricted in this way because it also interacts with non-TGM-containing sequences with a lower binding affinity (Lee, Seitz, et al., 2007). *TrmBL2* has no noticeable pattern with respect to other TGM-containing promoters (Lee, Seitz, et al., 2007; Lee, Surma, et al., 2007). The binding site of *TrmBL4*, however, has not yet been studied.

Because little is known about the metabolism of sugar and its regulation in *T. barophilus*, we investigated the potential role of *TrmBL4* through a reverse genetic approach in which its gene locus (*TERMP_00277*) was deleted. We then analysed the global secreted proteins, known as the ‘secretome’ of the wild-type (WT) and mutant strains at 0.1 MPa in the presence of different carbohydrates. This made it possible, using a liquid chromatography with tandem mass spectrometry (LC–MS/MS) proteomic approach, to reveal different secretomes linked to the presence or absence of *TrmBL4*. Finally, we assessed the potential of *TrmBL4* to regulate genes by performing reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments on target genes involved in *T. barophilus* sugar metabolism.

## EXPERIMENTAL PROCEDURES

### Construction of mutants: Pop-in/Pop-out technique

The *TERMP_00277* locus (*TrmBL4*) was deleted by the Pop-in/Pop-out method to generate the *T. barophilus* Δ*trmBL4* strain following the protocol previously published in Birien et al. (2018) and Thiel et al. (2014) (Table 1).

### Culture medium and growth experiments with sugar

Cells of *T. barophilus* WT *TbΔ517* (UBOCC-M-3300; Birien et al., 2018) and the derivative mutant *TbΔ517ΔtrmBL4* (UBOCC-M-3321) were cultivated under anaerobic conditions in Thermococcales rich medium (TRM; Zeng et al., 2009) at a 1× or 1.1× concentration (when sugars were added). The medium was supplemented with different sugar sources: maltose (maltose monohydrate, Sigma Lot no. SLCC3973), maltodextrin (maltodextrin, Sigma Lot no. MKCG8957), or pectin (pectin from citrus, Sigma Lot no. 97H0580) at a final concentration 0.5 g/L. The medium was then transferred into 50 mL penicillin vials with or without elemental sulphur (0.25 g/L), which acts as a terminal
<figure><img src="image_3.png" /></figure>

and *trmBL3* (*PF066*; Lee et al., 2008). Transcription assays show that *TrmB* of *P. furiosus* acts as a transcriptional repressor for the trehalose/maltose transport system (TM system) with high DNA binding affinity and for the MD system with lower affinity; this inhibition can be removed by the addition of specific sugars (Lee et al., 2005). Although, the *TrmBL1* of *P. furiosus* acts as a repressor of genes of the MD system and glycolytic enzymes, and as an activator of genes involved in glucogenic pathways, it is inactivated by maltose and maltotriose (Lee, Surma, et al., 2007). Only preliminary experiments have been done on *TrmBL2*, which appears to recognize TM and MD promoters and to play a role at the DNA level, clumping it into dense fibrous structures (Maruyama et al., 2011). The *TrmB* family performs a multiplicity of regulations by acting on the same promoters: at least three regulators, *TrmB*, *TrmBL1*, and *TrmBL2*, regulate gene expression at the level of the MD promoter in *P. furiosus* (Lee et al., 2008). Interestingly, this species can grow on carbohydrates such as cellobiose, maltose, and starch (Kengen et al., 1993; Schmid et al., 2013), which implies the secretion of enzymes such as amylopullulanase (Schmid et al., 2013) and an α-glycosidase (Constantino et al., 1990).

The *trmB* gene in *Thermococcus litoralis* was characterized as a repressor of the TM system (Lee et al., 2003). The role of the other regulators (*TrmBL3*, *TrmBL4*, and *TrmBL5*) in the Thermococcales (Gindner et al., 2014) remains unclear. *Thermococcus barophilus* MP, the first true hyperthermophilic piezophile archaeon isolated from a deep-sea hydrothermal vent (Marteinsson et al., 1999), possesses the *TERMP_01972* gene encoding *TrmB*, which might regulate the adjunct operon *TERMP_(01968-01973*; TM system); *TrmBL2* encoded by the *TERMP_00762* gene; *TrmBL4* encoded by the *TERMP_00277* gene, which can regulate the operon *TERMP_(00278-00281*) in the vicinity and divergently oriented (Figure 1A); *TrmBL1* encoded by the *TERMP_01652* gene, which could regulate the operon *TERMP_(01653-01658*; MD system; Figure 1B); and *TrmBL5* encoded by the *TERMP_01835* gene, which might be involved in the regulation of the *TERMP_(01835-01840*) cluster in *T. barophilus* (Figure 1C; Kim et al., 2016).

The binding sites of *TrmB* and *TrmBL1* were identified in *P. furiosus* (*TrmB*: TACTN₃AGTA for the TM system and TACT for the MD system; *TrmBL1*: TATCACN₅GTGATA for the MD system) as was the binding site of *TrmB* in *T. litoralis* (TACTN₃AGTA for the TM system; Lee et al., 2005). In 2006, Van de Werken et al. identified a conserved palindromic sequence, the Thermococcales glycolytic motif (TGM), TATCACN₅GTGATA, present upstream of all genes involved in sugar metabolism in several Thermococcales species as well as in the promoter region of the

MD operon, but not in the TM operon (Van de Werken et al., 2006). It has been shown that *TrmB* preferentially recognizes the TGM sequence (Lee, Seitz, et al., 2007), while *TrmBL1* target binding is not restricted in this way because it also interacts with non-TGM-containing sequences with a lower binding affinity (Lee, Seitz, et al., 2007). *TrmBL2* has no noticeable pattern with respect to other TGM-containing promoters (Lee, Seitz, et al., 2007; Lee, Surma, et al., 2007). The binding site of *TrmBL4*, however, has not yet been studied.

Because little is known about the metabolism of sugar and its regulation in *T. barophilus*, we investigated the potential role of *TrmBL4* through a reverse genetic approach in which its gene locus (*TERMP_00277*) was deleted. We then analysed the global secreted proteins, known as the ‘secretome’ of the wild-type (WT) and mutant strains at 0.1 MPa in the presence of different carbohydrates. This made it possible, using a liquid chromatography with tandem mass spectrometry (LC–MS/MS) proteomic approach, to reveal different secretomes linked to the presence or absence of *TrmBL4*. Finally, we assessed the potential of *TrmBL4* to regulate genes by performing reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments on target genes involved in *T. barophilus* sugar metabolism.

## EXPERIMENTAL PROCEDURES

### Construction of mutants: Pop-in/Pop-out technique

The *TERMP_00277* locus (*TrmBL4*) was deleted by the Pop-in/Pop-out method to generate the *T. barophilus* Δ*trmBL4* strain following the protocol previously published in Birien et al. (2018) and Thiel et al. (2014) (Table 1).

### Culture medium and growth experiments with sugar

Cells of *T. barophilus* WT *TbΔ517* (UBOCC-M-3300; Birien et al., 2018) and the derivative mutant *TbΔ517ΔtrmBL4* (UBOCC-M-3321) were cultivated under anaerobic conditions in Thermococcales rich medium (TRM; Zeng et al., 2009) at a 1× or 1.1× concentration (when sugars were added). The medium was supplemented with different sugar sources: maltose (maltose monohydrate, Sigma Lot no. SLCC3973), maltodextrin (maltodextrin, Sigma Lot no. MKCG8957), or pectin (pectin from citrus, Sigma Lot no. 97H0580) at a final concentration 0.5 g/L. The medium was then transferred into 50 mL penicillin vials with or without elemental sulphur (0.25 g/L), which acts as a terminal
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>FIGURE 1 The organization of three clusters encoding genes involved in sugar metabolism and upregulated by high hydrostatic pressure in *Thermococcus barophilus*. (A) The *TERMMP_(00275–00281)* cluster. (B) The *TERMMP_(01652–01658)* cluster (MD system). (C) The *TERMMP_(01835–01840)* cluster. The genes of interest representative of the target clusters for the reverse transcription quantitative polymerase chain reaction are highlighted in green. Red arrows indicate the TrmBL4 binding site and bold letters in the corresponding binding motifs indicate the non-conserved bases. Mal/Max indicates maltose/maltodextrin.</figcaption></figure>

electron acceptor that enhances the growth of Thermo-
coccales (Marteinsson et al., 1997). The cells were cul-
tured at 85°C under P_atm. To determine growth in each
treatment, cells were counted using a Thoma counting
chamber (0.01 mm depth, Weber, England) at ×100
magnification (Olympus BX40).

Recovery and concentration of
extracellular proteins by ultrafiltration

To recover the secretome, the WT, and the mutant cells
were cultured in 1-L bottles with 330 mL TRM medium
with and without sugars and then incubated at 85°C
under P_atm with shaking at 200 rpm. As noted above,
each of the three sources of sugar used was added to
give a final concentration of 0.5 g/L except for the pec-
tin, for which this was 0.15 g/L, because pectin

interferes with the good separation and visualization of
proteins. This minimum concentration of pectin was
determined after several kinetic studies. For this,
growth tracking of the cells was carried out in TRM
medium in the presence of different concentrations of
pectin (0.1, 0.15, 0.2, 0.25, 0.3, and 0.5 g/L) at 85°C
(data not shown). Sulphur was added (0.25 g/L) to each
treatment to enhance cell growth and thus obtain a suf-
ficient cell concentration to study the secretome of the
cells. The protocol for the concentration of extracellular
proteins was modified from Schmid et al. (2013). The
WT and mutant derivative cultures were collected at
the late exponential phase (1–2 × 10⁸ cells/mL) and
transferred to 450-mL centrifuge beakers to be centri-
fuged for 5 min at 200 × g, 4°C, to remove the rest of
the sulphur from the medium. The media were trans-
ferred to new beakers for further centrifugation for 1 h
at 8000 × g, 4°C. The supernatants were then filtered
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>FIGURE 1 The organization of three clusters encoding genes involved in sugar metabolism and upregulated by high hydrostatic pressure in *Thermococcus barophilus*. (A) The *TERMMP_(00275–00281)* cluster. (B) The *TERMMP_(01652–01658)* cluster (MD system). (C) The *TERMMP_(01835–01840)* cluster. The genes of interest representative of the target clusters for the reverse transcription quantitative polymerase chain reaction are highlighted in green. Red arrows indicate the TrmBL4 binding site and bold letters in the corresponding binding motifs indicate the non-conserved bases. Mal/Max indicates maltose/maltodextrin.</figcaption></figure>

electron acceptor that enhances the growth of Thermo-
coccales (Marteinsson et al., 1997). The cells were cul-
tured at 85°C under P_atm. To determine growth in each
treatment, cells were counted using a Thoma counting
chamber (0.01 mm depth, Weber, England) at ×100
magnification (Olympus BX40).

Recovery and concentration of
extracellular proteins by ultrafiltration

To recover the secretome, the WT, and the mutant cells
were cultured in 1-L bottles with 330 mL TRM medium
with and without sugars and then incubated at 85°C
under P_atm with shaking at 200 rpm. As noted above,
each of the three sources of sugar used was added to
give a final concentration of 0.5 g/L except for the pec-
tin, for which this was 0.15 g/L, because pectin

interferes with the good separation and visualization of
proteins. This minimum concentration of pectin was
determined after several kinetic studies. For this,
growth tracking of the cells was carried out in TRM
medium in the presence of different concentrations of
pectin (0.1, 0.15, 0.2, 0.25, 0.3, and 0.5 g/L) at 85°C
(data not shown). Sulphur was added (0.25 g/L) to each
treatment to enhance cell growth and thus obtain a suf-
ficient cell concentration to study the secretome of the
cells. The protocol for the concentration of extracellular
proteins was modified from Schmid et al. (2013). The
WT and mutant derivative cultures were collected at
the late exponential phase (1–2 × 10⁸ cells/mL) and
transferred to 450-mL centrifuge beakers to be centri-
fuged for 5 min at 200 × g, 4°C, to remove the rest of
the sulphur from the medium. The media were trans-
ferred to new beakers for further centrifugation for 1 h
at 8000 × g, 4°C. The supernatants were then filtered
<figure><img src="image_7.png" /></figure>

<table><caption>TABLE 1 The list of specific primers used to delete the *trmBL4* gene using the Pop-in/Pop-out technique and to target genes of interest using the RT-qPCR technique.</caption><thead><tr><th>TrmBL4 primer</th><th>Primer sequence</th></tr></thead><tbody><tr><td>Kpnl-D-TrmBL4 1 Fwd</td><td>5'AAAAAAGGTACCGCTATCAGAGTTTTTAGAGTAGCATCTTCAATG 3'</td></tr><tr><td>D TrmBL4 1 Rev</td><td>5' TGCAGTGAGGGGTGGTTAATAAAAGGAGGGATCTCCTTGAAAGCA 3'</td></tr><tr><td>D TrmBL4 2 Fwd</td><td>5'GGAGATCCCTCCTTTTATTAACCACCCCTCACTGCATTATATTCT 3'</td></tr><tr><td>BgIII-D TrmBL4 2 Rev</td><td>5'AAAAAAAGATCTATCTCCAAGCCATCCAGCACCGACA 3'</td></tr><tr><th>Target gene</th><th>Primer sequence</th></tr><tr><td>TERMP_00276 Fwd</td><td>5'ATTGCCTCATTGTTAATGCCGTATTCTCC 3'</td></tr><tr><td>TERMP_00276 Rev</td><td>5' TTAATTATGTCTCCGGTAAGCCGCG 3'</td></tr><tr><td>TERMP_00278 Fwd</td><td>5' CCACCTACAACAGTGACAACAACAAA 3'</td></tr><tr><td>TERMP_00278 Rev</td><td>5' GGGGAATTACCTCATATTTAACGCCGA 3'</td></tr><tr><td>TERMP_01652 Fwd</td><td>5' GCTTAAGCAGGGTTATCAGCGTTCT 3'</td></tr><tr><td>TERMP_01652 Rev</td><td>5' TTGGAGCCCTTCCAAGGGATAGA 3'</td></tr><tr><td>TERMP_01654 Fwd</td><td>5 GGAGGCGGAACAACCACTC 3'</td></tr><tr><td>TERMP_01654 Rev</td><td>5' GATTCAAAAAACCTGCGCCTCATTTG 3'</td></tr><tr><td>TERMP_01835 Fwd</td><td>5' CCTTCAATCTCTACAGGCTCCTTGC 3'</td></tr><tr><td>TERMP_01835 Rev</td><td>5' TTGCTGTTCATGTTTGACAGGTACTTCT 3'</td></tr><tr><td>TERMP_00342 (pcna) Fwd</td><td>5' GCATGAGGGCAATGGATCCA 3'</td></tr><tr><td>TERMP_00342 (pcna) Rev</td><td>5' GGGTTACCTCAAGGAAGTTCTCCTCA 3'</td></tr><tr><td>TERMP_00128 (30S) Fwd</td><td>5' GGCATAAACTTTGCCACGATGGTG 3'</td></tr><tr><td>TERMP_00128 (30S) Rev</td><td>5' TGCACCAATGAGGTGCATGTC 3'</td></tr></tbody></table>

Note: TERMP_00276 and TERMP_00278 representative of the TERMP_(00275-00281) cluster; TERMP_01652 representative of the TERMP_(01652-01658) cluster; TERMP_01654 representative of TERMP_(01652-01658); TERMP_01835 representative of TERMP_(01835-01840); TERMP_00342 and TERMP_00128 used as control genes.

Abbreviation: RT-qPCR, reverse transcription quantitative polymerase chain reaction.

on sterile filters (0.22-µm pore size) to remove cell remains. The proteins in the supernatant fractions (350 mL) were concentrated by two ultrafiltration procedures; first, by using an Amicon Ultra 15 10 K ultrafiltration unit following the manufacturer's procedure (Merck Millipore-Amicon® Ultra-15 10 K Centrifugal Filter Devices) to obtain a final volume of 100 µL. A minimum molecular weight of 10 kDa was chosen after research carried out in silico showed that most of the proteins likely to be secreted by *T. barophilus* would be larger than 10 kDa. The second ultrafiltration procedure was performed using an Amicon ultra 2-mL ultrafiltration unit. This filtration reduced the volume to 70 µL. To properly visualize proteins on an SDS-PAGE gel, the concentration of the proteins present in the supernatant must be 1 mg/mL; therefore, the protein concentration in each treatment was determined by Bradford assay using a standard range of known concentrations (Schmid et al., 2013). Additionally, to correctly filter the potentially secreted proteins, the cytosolic proteins were also concentrated. For this, the recovered cell pellet was centrifuged at 8000 × g for 30 min at 4°C. Cell lysis was then performed by sonication by pulses of 0.5/0.5 s for 5 min at 4°C. Afterward, the sonicate was centrifuged at 10,000 × g for 30 min. The same protocol was applied to concentrate the cytosolic proteins used as a control.

## SDS-PAGE and identification of proteins by MS

To visualize and separate proteins, the concentrated proteins from the supernatants of *T. barophilus* WT and mutant strains grown in the presence of sugars (maltose, maltodextrin, or pectin), were applied to a 4%-20% Tris-Glycine Mini-Protean gel (Bio-Rad, Hercules, California). The deposited protein volume ranged from 10 to 20 µL, containing 1 mg/mL of proteins, although the final deposited volume needed to be the same for all tested conditions. To cater for this, phosphate-buffered saline was used as an equilibration buffer and 4× XT sample buffer (Bio-Rad no. 1610791) was used as a stain. The gel was stained with Coomassie Brilliant Blue R250 (0.2% wt/vol, 40% vol/vol isopropanol, and 7% vol/vol acetic acid). The gel was then destained with a mixture of 300 mL 97% ethanol and 100 mL glacial acetic acid in a total volume of 1 L. Gel lanes were sliced into pieces and sent to the PAPPSO platform (Plateforme d'Analyse Protéomique de Paris Sud Ouest) for LC-MS/MS analysis on a 1D gel track (shotgun); the cytosolic concentrated proteins were rather used as a control for the secretion process. The gel slices were digested with 200 ng trypsin, then the samples were dispersed in 20 µL loading buffer and 4 µL were injected on a Fusion Lumos Orbitrap Tribrid mass
<figure><img src="image_6.png" /></figure>

<table><caption>TABLE 1 The list of specific primers used to delete the *trmBL4* gene using the Pop-in/Pop-out technique and to target genes of interest using the RT-qPCR technique.</caption><thead><tr><th>TrmBL4 primer</th><th>Primer sequence</th></tr></thead><tbody><tr><td>Kpnl-D-TrmBL4 1 Fwd</td><td>5'AAAAAAGGTACCGCTATCAGAGTTTTTAGAGTAGCATCTTCAATG 3'</td></tr><tr><td>D TrmBL4 1 Rev</td><td>5' TGCAGTGAGGGGTGGTTAATAAAAGGAGGGATCTCCTTGAAAGCA 3'</td></tr><tr><td>D TrmBL4 2 Fwd</td><td>5'GGAGATCCCTCCTTTTATTAACCACCCCTCACTGCATTATATTCT 3'</td></tr><tr><td>BgIII-D TrmBL4 2 Rev</td><td>5'AAAAAAAGATCTATCTCCAAGCCATCCAGCACCGACA 3'</td></tr><tr><th>Target gene</th><th>Primer sequence</th></tr><tr><td>TERMP_00276 Fwd</td><td>5'ATTGCCTCATTGTTAATGCCGTATTCTCC 3'</td></tr><tr><td>TERMP_00276 Rev</td><td>5' TTAATTATGTCTCCGGTAAGCCGCG 3'</td></tr><tr><td>TERMP_00278 Fwd</td><td>5' CCACCTACAACAGTGACAACAACAAA 3'</td></tr><tr><td>TERMP_00278 Rev</td><td>5' GGGGAATTACCTCATATTTAACGCCGA 3'</td></tr><tr><td>TERMP_01652 Fwd</td><td>5' GCTTAAGCAGGGTTATCAGCGTTCT 3'</td></tr><tr><td>TERMP_01652 Rev</td><td>5' TTGGAGCCCTTCCAAGGGATAGA 3'</td></tr><tr><td>TERMP_01654 Fwd</td><td>5 GGAGGCGGAACAACCACTC 3'</td></tr><tr><td>TERMP_01654 Rev</td><td>5' GATTCAAAAAACCTGCGCCTCATTTG 3'</td></tr><tr><td>TERMP_01835 Fwd</td><td>5' CCTTCAATCTCTACAGGCTCCTTGC 3'</td></tr><tr><td>TERMP_01835 Rev</td><td>5' TTGCTGTTCATGTTTGACAGGTACTTCT 3'</td></tr><tr><td>TERMP_00342 (pcna) Fwd</td><td>5' GCATGAGGGCAATGGATCCA 3'</td></tr><tr><td>TERMP_00342 (pcna) Rev</td><td>5' GGGTTACCTCAAGGAAGTTCTCCTCA 3'</td></tr><tr><td>TERMP_00128 (30S) Fwd</td><td>5' GGCATAAACTTTGCCACGATGGTG 3'</td></tr><tr><td>TERMP_00128 (30S) Rev</td><td>5' TGCACCAATGAGGTGCATGTC 3'</td></tr></tbody></table>

Note: TERMP_00276 and TERMP_00278 representative of the TERMP_(00275-00281) cluster; TERMP_01652 representative of the TERMP_(01652-01658) cluster; TERMP_01654 representative of TERMP_(01652-01658); TERMP_01835 representative of TERMP_(01835-01840); TERMP_00342 and TERMP_00128 used as control genes.

Abbreviation: RT-qPCR, reverse transcription quantitative polymerase chain reaction.

on sterile filters (0.22-µm pore size) to remove cell remains. The proteins in the supernatant fractions (350 mL) were concentrated by two ultrafiltration procedures; first, by using an Amicon Ultra 15 10 K ultrafiltration unit following the manufacturer's procedure (Merck Millipore-Amicon® Ultra-15 10 K Centrifugal Filter Devices) to obtain a final volume of 100 µL. A minimum molecular weight of 10 kDa was chosen after research carried out in silico showed that most of the proteins likely to be secreted by *T. barophilus* would be larger than 10 kDa. The second ultrafiltration procedure was performed using an Amicon ultra 2-mL ultrafiltration unit. This filtration reduced the volume to 70 µL. To properly visualize proteins on an SDS-PAGE gel, the concentration of the proteins present in the supernatant must be 1 mg/mL; therefore, the protein concentration in each treatment was determined by Bradford assay using a standard range of known concentrations (Schmid et al., 2013). Additionally, to correctly filter the potentially secreted proteins, the cytosolic proteins were also concentrated. For this, the recovered cell pellet was centrifuged at 8000 × g for 30 min at 4°C. Cell lysis was then performed by sonication by pulses of 0.5/0.5 s for 5 min at 4°C. Afterward, the sonicate was centrifuged at 10,000 × g for 30 min. The same protocol was applied to concentrate the cytosolic proteins used as a control.

## SDS-PAGE and identification of proteins by MS

To visualize and separate proteins, the concentrated proteins from the supernatants of *T. barophilus* WT and mutant strains grown in the presence of sugars (maltose, maltodextrin, or pectin), were applied to a 4%-20% Tris-Glycine Mini-Protean gel (Bio-Rad, Hercules, California). The deposited protein volume ranged from 10 to 20 µL, containing 1 mg/mL of proteins, although the final deposited volume needed to be the same for all tested conditions. To cater for this, phosphate-buffered saline was used as an equilibration buffer and 4× XT sample buffer (Bio-Rad no. 1610791) was used as a stain. The gel was stained with Coomassie Brilliant Blue R250 (0.2% wt/vol, 40% vol/vol isopropanol, and 7% vol/vol acetic acid). The gel was then destained with a mixture of 300 mL 97% ethanol and 100 mL glacial acetic acid in a total volume of 1 L. Gel lanes were sliced into pieces and sent to the PAPPSO platform (Plateforme d'Analyse Protéomique de Paris Sud Ouest) for LC-MS/MS analysis on a 1D gel track (shotgun); the cytosolic concentrated proteins were rather used as a control for the secretion process. The gel slices were digested with 200 ng trypsin, then the samples were dispersed in 20 µL loading buffer and 4 µL were injected on a Fusion Lumos Orbitrap Tribrid mass
<figure><img src="image_8.png" /></figure>

spectrometer. Quantitation of the proteins in all samples was performed using a Nanodrop 8000. Data processing was done with the X!TandemPipeline (0.4.40).

## Analysis of secreted proteins

To properly filter the potentially secreted proteins, two procedures were used. The first of these was the detection of signal peptides in silico with the OutCyte 1.0 program (Zhao et al., 2019). The second was the determination of the probability that a protein is secreted or not based on the results of MS analysis of the cytosolic proteins used as a control of secretion. In the calculations, detailed in Data S2, the determination of the secretion probability is based on the ratio of the amount of the protein in the supernatant to its presence in the cytosol while the cells are grown in the presence of the three tested sugars. Consequently, the secretion probability for each protein was determined and a heatmap was constructed based on a colorimetric scale showing the difference of normalized spectra (at 10,000/sample), which intuitively indicates the proteins that are the most abundant in the secretome of the tested samples. Hypothetical proteins with putative domain annotations were analysed using the Pfam 34.0 (Mistry et al., 2021) wrapper around the HMMER package, which analyses biosequences using hidden Markov models (Durbin et al., 1998), and the UCSC Archaeal Genome Browser database (Schneider et al., 2006). In the latter, the basic gene annotation is derived from the NCBI GenBank/RefSeq entries, with conservation overlays of sequences across multiple species and nucleotide/protein motifs, as well as non-coding RNA and operon predictions.

The hierarchically clustered heatmap was constructed with the cluster map API of the statistical data visualization Python library `seaborn` (Waskom, 2021). The weighted linkage method was used for calculating clusters.

## Sample RNA extraction and complementary DNA synthesis

RNA was extracted with TRIzol. For this, the strains were pre-cultured in a 20 mL TRM culture medium in the presence of sulphur, with and without maltose. WT and Δ*trmBL4* were inoculated at a rate of $2 \times 10^6$ cells/mL of fresh *T. barophilus* pre-culture, cultured for 6 h at 85°C at $P_{atm}$, and harvested at the mid-log phase by centrifugation at $5000 \times g$ for 6 min at 4°C. The pellet was re-suspended in 1 mL of Trizol and then transferred into 2 mL RNAse-free tubes. Then, 200 µL chloroform was added, followed by shaking by hand. The mixture was left for 3 min at room temperature. 15-min centrifugation was carried out at $12,000 \times g$, 4°C.

Afterward, the aqueous phase was placed in a 1.5 mL RNAse-free Eppendorf. To precipitate the RNA, 500 µL of isopropanol was added and the mixture incubated for 5 min at room temperature. The pellet was washed twice with 70% RNAse-free ethanol followed by centrifugation for 15 min at $12,000 \times g$. After successive washings, the supernatant was removed, the residual ethanol dried out, and the RNA dissolved in water. Residual DNA was removed by DNase treatment (3 U of Turbo™ DNase, Invitrogen, according to the manufacturer's instructions). The quantity of extracted RNA was determined using a Nanodrop 8000 (Thermo Fisher Scientific Inc.) and it was stored at -80°C until use. The reverse transcriptions were performed using the iScript Reverse Transcription Supermix Kit for RT-qPCR (Bio-Rad) to obtain complementary DNA (cDNA) concentration of ~2.5 ng/µL. cDNAs were amplified (5 min at 25°C, 20 min at 46°C, and 1 min at 95°C).

## Reverse transcription quantitative polymerase chain reaction

Quantitative PCR analysis was carried out on three clusters of genes, each with a representative gene, and primers were designed using SnapGene software (Figure 1 and Table 1). qPCR reactions with *pcna* and the *30S* gene were used as an internal control. Three biological replicates were performed for each treatment. Data were recorded and processed using Bio-Rad CFX Maestro software. The analysis was performed using the ΔΔCt method to calculate expression changes compared with the control (Livak & Schmittgen, 2001; Schmittgen & Zakrajsek, 2000). The control gene corresponds to a gene in the genome expressed under all growth conditions and is, therefore, unregulated. This means that the expression of this gene should be relatively stable under all the studied conditions.

## RESULTS

### *Thermococcus barophilus* growth enhancement by carbohydrates

To test the ability of *T. barophilus* to metabolize carbohydrates, we assessed its growth kinetics in TRM medium with or without the sugars maltose (MAL), maltodextrin (MAX), or pectin (PEC), each at a final concentration of 0.5 g/L. We compared the growth of the derivative mutant Δ*trmBL4* strain against the WT to explore the potential role of the uncharacterized TrmB-like family regulator. Knowing the critical role of sulphur in Thermococcales growth (Bertoldo & Antranikian, 2006), the cultures were also grown in the presence (Figure 2AB) or absence | (Figure 2C,D) of elemental sulphur (0.25 g/L).
<figure><img src="image_7.png" /></figure>

spectrometer. Quantitation of the proteins in all samples was performed using a Nanodrop 8000. Data processing was done with the X!TandemPipeline (0.4.40).

## Analysis of secreted proteins

To properly filter the potentially secreted proteins, two procedures were used. The first of these was the detection of signal peptides in silico with the OutCyte 1.0 program (Zhao et al., 2019). The second was the determination of the probability that a protein is secreted or not based on the results of MS analysis of the cytosolic proteins used as a control of secretion. In the calculations, detailed in Data S2, the determination of the secretion probability is based on the ratio of the amount of the protein in the supernatant to its presence in the cytosol while the cells are grown in the presence of the three tested sugars. Consequently, the secretion probability for each protein was determined and a heatmap was constructed based on a colorimetric scale showing the difference of normalized spectra (at 10,000/sample), which intuitively indicates the proteins that are the most abundant in the secretome of the tested samples. Hypothetical proteins with putative domain annotations were analysed using the Pfam 34.0 (Mistry et al., 2021) wrapper around the HMMER package, which analyses biosequences using hidden Markov models (Durbin et al., 1998), and the UCSC Archaeal Genome Browser database (Schneider et al., 2006). In the latter, the basic gene annotation is derived from the NCBI GenBank/RefSeq entries, with conservation overlays of sequences across multiple species and nucleotide/protein motifs, as well as non-coding RNA and operon predictions.

The hierarchically clustered heatmap was constructed with the cluster map API of the statistical data visualization Python library `seaborn` (Waskom, 2021). The weighted linkage method was used for calculating clusters.

## Sample RNA extraction and complementary DNA synthesis

RNA was extracted with TRIzol. For this, the strains were pre-cultured in a 20 mL TRM culture medium in the presence of sulphur, with and without maltose. WT and Δ*trmBL4* were inoculated at a rate of 2 × 10⁶ cells/mL of fresh *T. barophilus* pre-culture, cultured for 6 h at 85°C at P_atm, and harvested at the mid-log phase by centrifugation at 5000 × g for 6 min at 4°C. The pellet was re-suspended in 1 mL of Trizol and then transferred into 2 mL RNAse-free tubes. Then, 200 µL chloroform was added, followed by shaking by hand. The mixture was left for 3 min at room temperature. 15-min centrifugation was carried out at 12,000 × g, 4°C.

Afterward, the aqueous phase was placed in a 1.5 mL RNAse-free Eppendorf. To precipitate the RNA, 500 µL of isopropanol was added and the mixture incubated for 5 min at room temperature. The pellet was washed twice with 70% RNAse-free ethanol followed by centrifugation for 15 min at 12,000 × g. After successive washings, the supernatant was removed, the residual ethanol dried out, and the RNA dissolved in water. Residual DNA was removed by DNase treatment (3 U of Turbo™ DNase, Invitrogen, according to the manufacturer's instructions). The quantity of extracted RNA was determined using a Nanodrop 8000 (Thermo Fisher Scientific Inc.) and it was stored at -80°C until use. The reverse transcriptions were performed using the iScript Reverse Transcription Supermix Kit for RT-qPCR (Bio-Rad) to obtain complementary DNA (cDNA) concentration of ~2.5 ng/µL. cDNAs were amplified (5 min at 25°C, 20 min at 46°C, and 1 min at 95°C).

## Reverse transcription quantitative polymerase chain reaction

Quantitative PCR analysis was carried out on three clusters of genes, each with a representative gene, and primers were designed using SnapGene software (Figure 1 and Table 1). qPCR reactions with *pcna* and the *30S* gene were used as an internal control. Three biological replicates were performed for each treatment. Data were recorded and processed using Bio-Rad CFX Maestro software. The analysis was performed using the ΔΔCt method to calculate expression changes compared with the control (Livak & Schmittgen, 2001; Schmittgen & Zakrajsek, 2000). The control gene corresponds to a gene in the genome expressed under all growth conditions and is, therefore, unregulated. This means that the expression of this gene should be relatively stable under all the studied conditions.

## RESULTS

### *Thermococcus barophilus* growth enhancement by carbohydrates

To test the ability of *T. barophilus* to metabolize carbohydrates, we assessed its growth kinetics in TRM medium with or without the sugars maltose (MAL), maltodextrin (MAX), or pectin (PEC), each at a final concentration of 0.5 g/L. We compared the growth of the derivative mutant Δ*trmBL4* strain against the WT to explore the potential role of the uncharacterized TrmB-like family regulator. Knowing the critical role of sulphur in Thermococcales growth (Bertoldo & Antranikian, 2006), the cultures were also grown in the presence (Figure 2AB) or absence | (Figure 2C,D) of elemental sulphur (0.25 g/L).
<figure><img src="image_9.png" /></figure>

Our results show that growth is slightly enhanced in the presence of any sugar source, with an increase in the number of the WT cells, reaching a cell yield of $1.6 \times 10^8$ cells/mL at stationary phase (Figure 2A, blue dots, with sulphur), and a growth rate of $0.79 h^{-1}$ with maltose (Figure 2E). This compares with $8 \times 10^7$ cells/mL (Figure 2A, red dots, with sulphur) and a growth rate of $0.59 h^{-1}$ in the absence of sugars. In the case of the $\Delta trmBL4$ cells, the cell yield reached $2.5 \times 10^8$ cells/mL at stationary phase (Figure 2B, blue dots, with sulphur), with a growth rate of $0.96 h^{-1}$ with maltose (Figure 2E), compared with $9.1 \times 10^7$ cells/mL (Figure 2B, black dots, with sulphur) and a growth rate of $0.74 h^{-1}$ without any sugar (Figure 2E). The difference in cell concentration between the treatments with and without sugars was more pronounced when sulphur was absent from the medium. Cell growth in the presence of sugars reached $4.3 \times 10^7$ and $7 \times 10^7$ cells/mL, with growth rates of 0.42 and $0.56 h^{-1}$, in WT and $\Delta trmBL4$ cells, respectively, in the presence of maltodextrin (Figure 2C,D, green dots, and Figure 2E without sulphur) compared with $\sim 6 \times 10^6$ cells/mL for both strains in the absence of sugars ($0.2 h^{-1}$ growth rate; Figure 2C,D, red/black dots and Figure 2E without sulphur). Furthermore, a boost in the growth of the mutant cells compared with the WT strain was detected in the presence of maltose and sulphur (blue dots on Figure 2A,B, respectively), as the cell yields reached $2.5 \times 10^8$ cells/mL in $\Delta trmBL4$ at stationary phase but only $1.6 \times 10^8$ cells/mL in WT. These improvements in cell growth suggest the secretion of enzymes capable of degrading carbohydrates in the culture medium.

## Extracellular proteins detected in the supernatant

Cells from the WT and $\Delta trmBL4$ strains were grown in TRM medium supplemented with carbohydrates (maltose 0.5 g/L, maltodextrin 0.5 g/L, or pectin 0.15 g/L), in the presence of sulphur (0.25 g/L), to obtain sufficient proteins for testing and analysis. Cells were harvested at the end of the exponential growth phase (10 h) and their supernatant fractions were collected. The concentrated proteins were separated by 1D-gel electrophoresis (Figure 3), cut into slices, and sent to the PAPPSO platform for MS analysis, as were the cytosolic proteins used as a control. There, the proteins were analysed using high-resolution LC-MS/MS. A few additional proteins were detected on the gel in the case where the WT and mutant cells were cultured in the presence of sugars (indicated on the gel, Figure 3). For example, a protein of 44 kDa molecular weight was present in the secretome of the WT cells cultured in the presence of any sugar but absent when no sugars were provided (Figure 3, Band III). Another protein, 120 kDa, was only detected in the secretome of the WT cells in the

presence of maltodextrin (Figure 3, Band II). In addition, a protein of 87 kDa was detected in the secretome of the mutant cells in the presence of sugars but was absent when none were provided (Figure 3, Band V). Despite the decrease in the concentration of pectin (0.15 g/L instead of 0.5 g/L) for the proteomic analysis, as explained in the experimental procedures section, technical issues were encountered when preparing proteins from the supernatant of the WT cells grown in the presence of pectin (data not shown). Consequently, the secretome of $\Delta trmBL4$ cells in the presence of pectin was not studied. To obtain a description of the dataset generated by the LC-MS/MS, a hierarchical clustering heatmap was constructed based on the values, showing the difference of normalized spectra (at 10,000/sample). This method reveals which of the secreted proteins are the most abundant in the secretome of the tested samples (Figure 4 and Data S3). The detected cytosolic proteins were removed from the overall data (Data S1) to focus on the secretome. A total of at least 33 proteins were secreted in the supernatant of the WT and mutant cells cultured in the presence of sugars (Figure 4, Table 2, and Data S2). The corresponding heatmap highlights a WT-mutant dichotomy. Figure 4 shows the mutant cells on the left and the WT cells on the right, cultivated in the presence or absence of sugars. With the added carbohydrates, we see a clear shift in the secreted proteins of the mutant cells, with only some slight differences between maltose and maltodextrin. This did not happen with the WT cells, where notable differences could be observed, for example, the protein encoded by TERMP_01807 (S-layer domain) was only expressed in the WT cells with maltodextrin. Interestingly, no common effect was detected between the three sugars, which could be because pectin may induce another secretome closer to the one that appears without sugar than to those with maltose or maltodextrin.

Among the proteins of the secretome, several were repressed in the mutant cells under particular conditions. These included a protein encoded by TERMP_01556, a pyruvate ferredoxin oxidoreductase $\Delta$-subunit catalysing one of the final steps in carbohydrate fermentation (Blamey & Adams, 1993), which was found to be repressed in the mutant cells regardless of sugar availability. Two hypothetical proteins, respectively encoded by TERMP_01372 and TERMP_02026, were also repressed in the mutant. Conversely, we detected an overexpression of some proteins in the mutant strain. These included the protein encoded by TERMP_02130, which was expressed slightly more in the mutant cells than in the WT cells; and the protein encoded by TERMP_00597, hypothetically coding for an alpha-galactosidase NEW3 domain, whose expression was higher in the mutant cells cultured without sugars. Additionally, the proteins encoded by
<figure><img src="image_8.png" /></figure>

Our results show that growth is slightly enhanced in the presence of any sugar source, with an increase in the number of the WT cells, reaching a cell yield of $1.6 \times 10^8$ cells/mL at stationary phase (Figure 2A, blue dots, with sulphur), and a growth rate of $0.79 h^{-1}$ with maltose (Figure 2E). This compares with $8 \times 10^7$ cells/mL (Figure 2A, red dots, with sulphur) and a growth rate of $0.59 h^{-1}$ in the absence of sugars. In the case of the $\Delta trmBL4$ cells, the cell yield reached $2.5 \times 10^8$ cells/mL at stationary phase (Figure 2B, blue dots, with sulphur), with a growth rate of $0.96 h^{-1}$ with maltose (Figure 2E), compared with $9.1 \times 10^7$ cells/mL (Figure 2B, black dots, with sulphur) and a growth rate of $0.74 h^{-1}$ without any sugar (Figure 2E). The difference in cell concentration between the treatments with and without sugars was more pronounced when sulphur was absent from the medium. Cell growth in the presence of sugars reached $4.3 \times 10^7$ and $7 \times 10^7$ cells/mL, with growth rates of 0.42 and $0.56 h^{-1}$, in WT and $\Delta trmBL4$ cells, respectively, in the presence of maltodextrin (Figure 2C,D, green dots, and Figure 2E without sulphur) compared with $\sim 6 \times 10^6$ cells/mL for both strains in the absence of sugars ($0.2 h^{-1}$ growth rate; Figure 2C,D, red/black dots and Figure 2E without sulphur). Furthermore, a boost in the growth of the mutant cells compared with the WT strain was detected in the presence of maltose and sulphur (blue dots on Figure 2A,B, respectively), as the cell yields reached $2.5 \times 10^8$ cells/mL in $\Delta trmBL4$ at stationary phase but only $1.6 \times 10^8$ cells/mL in WT. These improvements in cell growth suggest the secretion of enzymes capable of degrading carbohydrates in the culture medium.

## Extracellular proteins detected in the supernatant

Cells from the WT and $\Delta trmBL4$ strains were grown in TRM medium supplemented with carbohydrates (maltose 0.5 g/L, maltodextrin 0.5 g/L, or pectin 0.15 g/L), in the presence of sulphur (0.25 g/L), to obtain sufficient proteins for testing and analysis. Cells were harvested at the end of the exponential growth phase (10 h) and their supernatant fractions were collected. The concentrated proteins were separated by 1D-gel electrophoresis (Figure 3), cut into slices, and sent to the PAPPSO platform for MS analysis, as were the cytosolic proteins used as a control. There, the proteins were analysed using high-resolution LC-MS/MS. A few additional proteins were detected on the gel in the case where the WT and mutant cells were cultured in the presence of sugars (indicated on the gel, Figure 3). For example, a protein of 44 kDa molecular weight was present in the secretome of the WT cells cultured in the presence of any sugar but absent when no sugars were provided (Figure 3, Band III). Another protein, 120 kDa, was only detected in the secretome of the WT cells in the

presence of maltodextrin (Figure 3, Band II). In addition, a protein of 87 kDa was detected in the secretome of the mutant cells in the presence of sugars but was absent when none were provided (Figure 3, Band V). Despite the decrease in the concentration of pectin (0.15 g/L instead of 0.5 g/L) for the proteomic analysis, as explained in the experimental procedures section, technical issues were encountered when preparing proteins from the supernatant of the WT cells grown in the presence of pectin (data not shown). Consequently, the secretome of $\Delta trmBL4$ cells in the presence of pectin was not studied. To obtain a description of the dataset generated by the LC-MS/MS, a hierarchical clustering heatmap was constructed based on the values, showing the difference of normalized spectra (at 10,000/sample). This method reveals which of the secreted proteins are the most abundant in the secretome of the tested samples (Figure 4 and Data S3). The detected cytosolic proteins were removed from the overall data (Data S1) to focus on the secretome. A total of at least 33 proteins were secreted in the supernatant of the WT and mutant cells cultured in the presence of sugars (Figure 4, Table 2, and Data S2). The corresponding heatmap highlights a WT-mutant dichotomy. Figure 4 shows the mutant cells on the left and the WT cells on the right, cultivated in the presence or absence of sugars. With the added carbohydrates, we see a clear shift in the secreted proteins of the mutant cells, with only some slight differences between maltose and maltodextrin. This did not happen with the WT cells, where notable differences could be observed, for example, the protein encoded by TERMP_01807 (S-layer domain) was only expressed in the WT cells with maltodextrin. Interestingly, no common effect was detected between the three sugars, which could be because pectin may induce another secretome closer to the one that appears without sugar than to those with maltose or maltodextrin.

Among the proteins of the secretome, several were repressed in the mutant cells under particular conditions. These included a protein encoded by TERMP_01556, a pyruvate ferredoxin oxidoreductase $\Delta$-subunit catalysing one of the final steps in carbohydrate fermentation (Blamey & Adams, 1993), which was found to be repressed in the mutant cells regardless of sugar availability. Two hypothetical proteins, respectively encoded by TERMP_01372 and TERMP_02026, were also repressed in the mutant. Conversely, we detected an overexpression of some proteins in the mutant strain. These included the protein encoded by TERMP_02130, which was expressed slightly more in the mutant cells than in the WT cells; and the protein encoded by TERMP_00597, hypothetically coding for an alpha-galactosidase NEW3 domain, whose expression was higher in the mutant cells cultured without sugars. Additionally, the proteins encoded by
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>FIG URE 2 Tracking the growth of the wild-type (WT) and the Δ*trmBL4* cells with or without sugars in the presence and absence of sulphur at 0.1 MPa. WT cells cultured without sugars (●); Δ*trmBL4* cells cultured without sugars (●); cells cultured in the presence of maltose (●), maltodextrin (●), or pectin (●). (A) The growth of the WT cells in the presence and absence of sugars in the presence of sulphur. (B) The growth of Δ*trmBL4* cells in the presence and absence of sugars in the presence of sulphur. (C) The growth of the WT cells in the presence and absence of sugars in the absence of sulphur. (D) The growth of Δ*trmBL4* cells in the presence and absence of sugars in the absence of sulphur. (E) Comparison of the growth rate of the WT and Δ*trmBL4* cells in the presence of maltose (Mal), maltodextrin (Max), and pectin (Pec), in the presence and absence of sulphur at 0.1 MPa. A different pattern is used for each type of sugar for both WT and Δ*trmBL4* cells. A solid fill is used where no sugar was added. The growth rate, which is the inverse of generation time (G), represents the number of generations per unit of time in an exponentially growing culture. It is equal to 1/G, expressed in inverse units of time (h⁻¹). The generation time (G) represents the time necessary for the doubling of a population in the exponential phase. Error bars represent standard deviations obtained from at least three biological replicates with three technical replicates each.</figcaption></figure>

TERMP_00275, TERMP_00281, and TERMP_00278, which are part of the HHP-responsive cluster and close to the TrmBL4 regulator (Vannier et al., 2015), were also overexpressed and only detected in the case of the mutant. However, the protein encoded by TERMP_01657, coding for an amylopullulanase that is part of the second HHP-responsive cluster TERMP_

(01653–01658; MD system), was overexpressed in both the WT and mutant secretomes in the presence of maltose and maltodextrin, whereas the other proteins of this operon were only slightly overexpressed in the mutant in the presence of sugars (Data S1). Regarding the TERMP_(01836–01838) operon, no expression was detected.
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>FIG URE 2 Tracking the growth of the wild-type (WT) and the Δ*trmBL4* cells with or without sugars in the presence and absence of sulphur at 0.1 MPa. WT cells cultured without sugars (●); Δ*trmBL4* cells cultured without sugars (●); cells cultured in the presence of maltose (●), maltodextrin (●), or pectin (●). (A) The growth of the WT cells in the presence and absence of sugars in the presence of sulphur. (B) The growth of Δ*trmBL4* cells in the presence and absence of sugars in the presence of sulphur. (C) The growth of the WT cells in the presence and absence of sugars in the absence of sulphur. (D) The growth of Δ*trmBL4* cells in the presence and absence of sugars in the absence of sulphur. (E) Comparison of the growth rate of the WT and Δ*trmBL4* cells in the presence of maltose (Mal), maltodextrin (Max), and pectin (Pec), in the presence and absence of sulphur at 0.1 MPa. A different pattern is used for each type of sugar for both WT and Δ*trmBL4* cells. A solid fill is used where no sugar was added. The growth rate, which is the inverse of generation time (G), represents the number of generations per unit of time in an exponentially growing culture. It is equal to 1/G, expressed in inverse units of time (h⁻¹). The generation time (G) represents the time necessary for the doubling of a population in the exponential phase. Error bars represent standard deviations obtained from at least three biological replicates with three technical replicates each.</figcaption></figure>

TERMP_00275, TERMP_00281, and TERMP_00278, which are part of the HHP-responsive cluster and close to the TrmBL4 regulator (Vannier et al., 2015), were also overexpressed and only detected in the case of the mutant. However, the protein encoded by TERMP_01657, coding for an amylopullulanase that is part of the second HHP-responsive cluster TERMP_

(01653–01658; MD system), was overexpressed in both the WT and mutant secretomes in the presence of maltose and maltodextrin, whereas the other proteins of this operon were only slightly overexpressed in the mutant in the presence of sugars (Data S1). Regarding the TERMP_(01836–01838) operon, no expression was detected.
<figure><img src="image_12.png" /></figure>

FIG URE 3 A representative Coomassie blue stained SDS-PAGE gel showing concentrated proteins from *Thermococcus barophilus* wild-type (WT) and *ΔtrmBL4* cell culture supernatants. 1. PageRuler™ Prestained Protein Ladder; molecular weights are indicated in kDa; 1 mg/mL of proteins from each culture supernatant was loaded. 2. WT culture in the absence of sugars. 3. WT culture in the presence of maltose. 4. WT culture in the presence of maltodextrin. 5. WT culture in the presence of pectin. 6. *ΔtrmBL4* culture in the absence of sugars. 7. *ΔtrmBL4* culture in the presence of maltose. 8. *ΔtrmBL4* culture in the presence of maltodextrin. A few additional bands detected in the presence of sugars are indicated by Roman numerals.

Surprisingly, the proteins encoded by *TER MP_00360* and *TERMP_02126* were expressed in all tested conditions. The first of these, corresponding to DNA protection during starvation protein, is known to protect DNA during nutrient starvation and against oxidative DNA damage (Frenkiel-Krispin et al., 2001). The second corresponds to a vWFA domain-containing protein known to be involved in cell adhesion, signal transduction, extracellular matrix proteins, and integrin receptors (Whittaker & Hynes, 2002).

Furthermore, the expression of the hypothetical proteins encoded by *TERMP_01984*, *TERMP_01543*, and *TERMP_02023* was altered by both the mutation and the presence of sugars because they are expressed only in the WT cells in the absence of sugars. In contrast, we detected the presence of a cyclomaltodextrin glucanotransferase, encoded by *TERMP_01455*, whose expression is controlled by the presence of maltose and maltodextrin in the medium independently of the mutation.

In addition, we observed that the expression of putative RNA binding proteins was altered the proteins encoded by *TERMP_00403* and *TERMP_01500* were repressed in the mutant cells compared with the WT cells in the absence of sugars. This was also the case with the phosphoesterase encoded by *TERMP_00076*, whose expression was activated in the mutant cells by the addition of sugars. These proteins were found in the supernatant of the cells but had no detectable signal peptide.

## Relative expression of genes in the presence and absence of sugars studied by RT-qPCR

To determine the potential consequences of the deletion of the *trmBL4* gene for the expression of the three HHP-responsive gene clusters involved in the metabolism of sugars in *T. barophilus*, we performed RT-qPCR experiments targeting some representative genes of clusters of interest determined by the results of the LC-MS/MS analysis and the transcriptomics data published by Vannier et al. (2015). These genes were *TERMP_00276* and *TERMP_00278*, representative of the *TERMP_(00278-00282)* cluster (Figure 1A); *TERMP_01652* and *TERMP_01654*, representative of the *TERMP_(01652-01658)* cluster (Figure 1B); and *TERMP_01835*, representative of the *TERMP_(01835-01840)* cluster (Figure 1C). The effects of the deletion of the *trmBL4* gene and of sugar addition were tested (Figure 5A,B, respectively). When comparing the results of *ΔtrmBL4* and WT cells cultivated in the absence of maltose (Figure 5A), a repression of the expression of the representative genes of *TERMP_(00275-00281)* and *TERMP_(01652-01658)* was observed in the mutant strain, implying that *trmBL4* acts as an activator of the expression of the corresponding genes in the absence of maltose. Moreover, the results of WT cells cultivated in the presence or absence of maltose highlight repression of the same representative genes in the presence of maltose (*TERMP_00276*, *TERMP_00278*, *TERMP_01652*, and *TERMP_01654*). Comparison between the results of *ΔtrmBL4* and WT showed that, in the presence of maltose, the expression of the representative genes of *TERMP_(00275-00281)* and *TERMP_(01652-01658)* was increased in the mutant strain, linking *TrmBL4* as a repressor of the gene expression. So far, the operon *TERMP_(00275-00282)* seemed to be regulated by *TrmBL4*. The deletion of the *trmBL4* gene once again allowed, in the presence of maltose, an increase in the relative expression of this operon (Figure 5B). Concerning the expression of *TERMP_01654*, the representative gene of the *TERMP_(01652-01658)* operon, the increase was smaller, probably because of a lower affinity of the *trmBL4* regulator on this operon. In contrast, the expression of *TERMP_01835*, the representative gene of the *TERMP_(01835-01840)* cluster, was not affected by *TrmBL4* and was only slightly impacted by the addition of sugar.

## Identification of TrmBL4 DNA binding domains

To check for a connection between *TrmBL4* and the genes that seem to be controlled by this regulator, an in silico screening for the *TrmB*-like binding motif was
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>FIG URE 3 A representative Coomassie blue stained SDS-PAGE gel showing concentrated proteins from *Thermococcus barophilus* wild-type (WT) and Δ*trmBL4* cell culture supernatants.</figcaption></figure>

1. PageRuler™ Prestained Protein Ladder; molecular weights are indicated in kDa; 1 mg/mL of proteins from each culture supernatant was loaded. 2. WT culture in the absence of sugars. 3. WT culture in the presence of maltose. 4. WT culture in the presence of maltodextrin. 5. WT culture in the presence of pectin. 6. Δ*trmBL4* culture in the absence of sugars. 7. Δ*trmBL4* culture in the presence of maltose. 8. Δ*trmBL4* culture in the presence of maltodextrin. A few additional bands detected in the presence of sugars are indicated by Roman numerals.

Surprisingly, the proteins encoded by *TER MP_00360* and *TERMP_02126* were expressed in all tested conditions. The first of these, corresponding to DNA protection during starvation protein, is known to protect DNA during nutrient starvation and against oxidative DNA damage (Frenkiel-Krispin et al., 2001). The second corresponds to a vWFA domain-containing protein known to be involved in cell adhesion, signal transduction, extracellular matrix proteins, and integrin receptors (Whittaker & Hynes, 2002).

Furthermore, the expression of the hypothetical proteins encoded by *TERMP_01984*, *TERMP_01543*, and *TERMP_02023* was altered by both the mutation and the presence of sugars because they are expressed only in the WT cells in the absence of sugars. In contrast, we detected the presence of a cyclomaltodextrin glucanotransferase, encoded by *TERMP_01455*, whose expression is controlled by the presence of maltose and maltodextrin in the medium independently of the mutation.

In addition, we observed that the expression of putative RNA binding proteins was altered the proteins encoded by *TERMP_00403* and *TERMP_01500* were repressed in the mutant cells compared with the WT cells in the absence of sugars. This was also the case with the phosphoesterase encoded by *TERMP_00076*, whose expression was activated in the mutant cells by the addition of sugars. These proteins were found in the supernatant of the cells but had no detectable signal peptide.

## Relative expression of genes in the presence and absence of sugars studied by RT-qPCR

To determine the potential consequences of the deletion of the *trmBL4* gene for the expression of the three HHP-responsive gene clusters involved in the metabolism of sugars in *T. barophilus*, we performed RT-qPCR experiments targeting some representative genes of clusters of interest determined by the results of the LC-MS/MS analysis and the transcriptomics data published by Vannier et al. (2015). These genes were *TERMP_00276* and *TERMP_00278*, representative of the *TERMP_(00278-00282)* cluster (Figure 1A); *TERMP_01652* and *TERMP_01654*, representative of the *TERMP_(01652-01658)* cluster (Figure 1B); and *TERMP_01835*, representative of the *TERMP_(01835-01840)* cluster (Figure 1C). The effects of the deletion of the *trmBL4* gene and of sugar addition were tested (Figure 5A,B, respectively). When comparing the results of *ΔtrmBL4* and WT cells cultivated in the absence of maltose (Figure 5A), a repression of the expression of the representative genes of *TERMP_(00275-00281)* and *TERMP_(01652-01658)* was observed in the mutant strain, implying that *trmBL4* acts as an activator of the expression of the corresponding genes in the absence of maltose. Moreover, the results of WT cells cultivated in the presence or absence of maltose highlight repression of the same representative genes in the presence of maltose (*TERMP_00276*, *TERMP_00278*, *TERMP_01652*, and *TERMP_01654*). Comparison between the results of *ΔtrmBL4* and WT showed that, in the presence of maltose, the expression of the representative genes of *TERMP_(00275-00281)* and *TERMP_(01652-01658)* was increased in the mutant strain, linking *TrmBL4* as a repressor of the gene expression. So far, the operon *TERMP_(00275-00282)* seemed to be regulated by *TrmBL4*. The deletion of the *trmBL4* gene once again allowed, in the presence of maltose, an increase in the relative expression of this operon (Figure 5B). Concerning the expression of *TERMP_01654*, the representative gene of the *TERMP_(01652-01658)* operon, the increase was smaller, probably because of a lower affinity of the *trmBL4* regulator on this operon. In contrast, the expression of *TERMP_01835*, the representative gene of the *TERMP_(01835-01840)* cluster, was not affected by *TrmBL4* and was only slightly impacted by the addition of sugar.

## Identification of TrmBL4 DNA binding domains

To check for a connection between *TrmBL4* and the genes that seem to be controlled by this regulator, an in silico screening for the *TrmB*-like binding motif was
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>FIG URE 4 Hierarchical heat map for the visualization of the differential expression of the secreted proteins in each sample. The heat map was produced with the seaborn program using the liquid chromatography with tandem mass spectrometry data and shows only the secreted proteins (Data S3) from Data S1. The samples are shown in the columns and the ordered loci for the genes encoding the secreted proteins in the rows. Columns are centred, with relative abundance (the difference of normalized spectra) represented by the darkness of shading (white: lower abundance; black: higher abundance) of the secreted proteins in the tested conditions. Mal, Max, and Pec indicate maltose, maltodextrin, and pectin, respectively.</figcaption></figure>

performed on the whole genome of *T. barophilus* based on the known binding motifs of TrmB and TrmBL1 discovered in other Thermococcales (Lee et al., 2008; Lee, Surma, et al., 2007). We detected a high degree of conservation between the binding motifs in *T. barophilus* compared with those known in *P. furiosus*. Concerning the *Pfu-TrmB* binding site, the same *TACTN<sub>3</sub>AGTA* sequence was found in *T. barophilus* for the TM system, except for two nucleotides that differ from the *Pfu-TrmBL1* binding site for the MD system (*TATCACN<sub>5</sub>GTGATA* in *P. furiosus* and *AATCATN<sub>5</sub>GTGATA* in *T. barophilus*).

A binding site (*AATCATN*<sub>5</sub>*GTGATA*) was found upstream of *TERMP*_01653 (MD system) at position 1,439,024–1,439,040. This binding motif includes the second half of the TGM sequence **GTGATA**. Moreover, the binding site (*TACTN*<sub>3</sub>*AGTA*) was detected upstream of *TERMP*_01968 (TM system) at the positions 1,762,048–1,762,068 and 1,762,230–1,762,240. Two additional binding sites (*TATTCTGAGTT*, pos. 232,136–232,146 and *TACTN*<sub>6</sub>*AGATA*, pos. 1,624,822–1,624,836) were detected upstream of *TERMP*_00278 and *TERMP*_01840. Three additional binding sites (*TACTTAGTT*, pos. 1,295,540–1,295,548; *TACTGCTT*,
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>FIG URE 4 Hierarchical heat map for the visualization of the differential expression of the secreted proteins in each sample. The heat map was produced with the seaborn program using the liquid chromatography with tandem mass spectrometry data and shows only the secreted proteins (Data S3) from Data S1. The samples are shown in the columns and the ordered loci for the genes encoding the secreted proteins in the rows. Columns are centred, with relative abundance (the difference of normalized spectra) represented by the darkness of shading (white: lower abundance; black: higher abundance) of the secreted proteins in the tested conditions. Mal, Max, and Pec indicate maltose, maltodextrin, and pectin, respectively.</figcaption></figure>

performed on the whole genome of *T. barophilus* based on the known binding motifs of TrmB and TrmBL1 discovered in other Thermococcales (Lee et al., 2008; Lee, Surma, et al., 2007). We detected a high degree of conservation between the binding motifs in *T. barophilus* compared with those known in *P. furiosus*. Concerning the *Pfu-TrmB* binding site, the same *TACTN<sub>3</sub>AGTA* sequence was found in *T. barophilus* for the TM system, except for two nucleotides that differ from the *Pfu-TrmBL1* binding site for the MD system (*TATCACN<sub>5</sub>GTGATA* in *P. furiosus* and *AATCATN<sub>5</sub>GTGATA* in *T. barophilus*).

A binding site (*AATCATN*<sub>5</sub>*GTGATA*) was found upstream of *TERMP*_01653 (MD system) at position 1,439,024–1,439,040. This binding motif includes the second half of the TGM sequence **GTGATA**. Moreover, the binding site (*TACTN*<sub>3</sub>*AGTA*) was detected upstream of *TERMP*_01968 (TM system) at the positions 1,762,048–1,762,068 and 1,762,230–1,762,240. Two additional binding sites (*TATTCTGAGTT*, pos. 232,136–232,146 and *TACTN*<sub>6</sub>*AGATA*, pos. 1,624,822–1,624,836) were detected upstream of *TERMP*_00278 and *TERMP*_01840. Three additional binding sites (*TACTTAGTT*, pos. 1,295,540–1,295,548; *TACTGCTT*,
<figure><img src="image_15.png" /></figure>

<table><caption>TABLE 2 The list of secreted proteins identified in the supernatant of *Thermococcus barophilus* wild-type and Δ*trmBL4* cells grown on sugars.</caption><thead><tr><th>Ordered locus</th><th>Found only in the supernatant</th><th>Secretion probability</th><th>N-terminal signal sequence<sup>a</sup></th><th>Molecular weight</th><th>Description</th><th>Pfam/UCSC archaeal genome<sup>b</sup></th><th>TrmBL4 binding motif<sup>c</sup></th></tr></thead><tbody><tr><td>TERMP_01807</td><td>Yes</td><td>0.5</td><td>Yes</td><td>101.5</td><td>S-layer domain</td><td></td><td>No</td></tr><tr><td>TERMP_01984</td><td>Yes</td><td>1</td><td>Yes</td><td>68.1</td><td>Lipoprotein</td><td></td><td>No</td></tr><tr><td>TERMP_01543</td><td>Yes</td><td>1</td><td>Yes</td><td>45.2</td><td>Uncharacterized protein</td><td>Eight-cysteine-cluster domain</td><td>No</td></tr><tr><td>TERMP_02023</td><td>Yes</td><td>1</td><td>Yes</td><td>22</td><td>Flagellin</td><td></td><td>Yes</td></tr><tr><td>TERMP_00515</td><td>Yes</td><td>0.5</td><td>Yes</td><td>70.5</td><td>S-layer_C domain-containing protein</td><td></td><td>No</td></tr><tr><td>TERMP_01415</td><td>Yes</td><td>0.5</td><td>Yes</td><td>71.6</td><td>Uncharacterized protein</td><td>Hypothetical protein</td><td>No</td></tr><tr><td>TERMP_01455</td><td>Yes</td><td>0.5</td><td>Yes</td><td>81</td><td>Cyclomaltodextrin glucanotransferase</td><td></td><td>No</td></tr><tr><td>TERMP_01372</td><td>Yes</td><td>1</td><td>Yes</td><td>56.4</td><td>Uncharacterized protein</td><td>Amylopullulanase</td><td>No</td></tr><tr><td>TERMP_02026</td><td>Yes</td><td>1</td><td>Yes</td><td>51</td><td>Uncharacterized protein</td><td>Hypothetical protein</td><td>No</td></tr><tr><td>TERMP_00403</td><td>Yes</td><td>1</td><td>No</td><td>8.9</td><td>RNA-binding protein</td><td></td><td>No</td></tr><tr><td>TERMP_01263</td><td>Yes</td><td>1</td><td>No</td><td>27.7</td><td>Metal-dependent hydrolase</td><td></td><td>No</td></tr><tr><td>TERMP_00076</td><td>Yes</td><td>1</td><td>No</td><td>18.8</td><td>Phosphoesterase</td><td></td><td>No</td></tr><tr><td>TERMP_01902</td><td>Yes</td><td>1</td><td>No</td><td>17.6</td><td>Peptidyl-prolyl cis-trans isomerase</td><td></td><td>No</td></tr><tr><td>TERMP_02030</td><td>Yes</td><td>1</td><td>No</td><td>22</td><td>GMP synthase</td><td></td><td>No</td></tr><tr><td>TERMP_02179</td><td>Yes</td><td>1</td><td>Yes</td><td>209.5</td><td>Peptidase_S8 domain-containing protein</td><td></td><td>No</td></tr><tr><td>TERMP_00275</td><td>Yes</td><td>0.5</td><td>No</td><td>86.8</td><td>Maltose phosphorylase</td><td></td><td>No</td></tr><tr><td>TERMP_00281</td><td>Yes</td><td>0.5</td><td>No</td><td>40.1</td><td>Sugar ABC transporter</td><td></td><td>No</td></tr><tr><td>TERMP_01500</td><td>Yes</td><td>1</td><td>No</td><td>8.2</td><td>RNA-binding protein</td><td></td><td>Yes</td></tr><tr><td>TERMP_00360</td><td>No</td><td>1</td><td>No</td><td>21.2</td><td>DNA protection during starvation protein</td><td></td><td>No</td></tr><tr><td>TERMP_00817</td><td>No</td><td>1</td><td>No</td><td>21.5</td><td>Thymidine kinase</td><td></td><td>No</td></tr><tr><td>TERMP_01559</td><td>No</td><td>1</td><td>No</td><td>12</td><td>Ketoisovalerate oxidoreductase subunit</td><td></td><td>No</td></tr><tr><td>TERMP_01449</td><td>Yes</td><td>1</td><td>Yes</td><td>76.4</td><td>Dockerin domain-containing protein</td><td></td><td>No</td></tr><tr><td>TERMP_01791</td><td>No</td><td>1</td><td>Yes</td><td>48.5</td><td>S-layer-like array protein</td><td></td><td>No</td></tr><tr><td>TERMP_00278</td><td>Yes</td><td>0.5</td><td>Yes</td><td>50.9</td><td>MalE-like Mal/Max ABC transporter</td><td></td><td>No</td></tr><tr><td>TERMP_02126</td><td>No</td><td>1</td><td>Yes</td><td>179.8</td><td>vWFA domain-containing protein</td><td></td><td>No</td></tr></tbody><tfoot><tr><td colspan="8" style="text-align: right;">(Continues)</td></tr></tfoot></table>
<figure><img src="image_15.png" /></figure>

<table><caption>TABLE 2 The list of secreted proteins identified in the supernatant of *Thermococcus barophilus* wild-type and Δ*trmBL4* cells grown on sugars.</caption><thead><tr><th>Ordered locus</th><th>Found only in the supernatant</th><th>Secretion probability</th><th>N-terminal signal sequence<sup>a</sup></th><th>Molecular weight</th><th>Description</th><th>Pfam/UCSC archaeal genome<sup>b</sup></th><th>TrmBL4 binding motif<sup>c</sup></th></tr></thead><tbody><tr><td>TERMP_01807</td><td>Yes</td><td>0.5</td><td>Yes</td><td>101.5</td><td>S-layer domain</td><td></td><td>No</td></tr><tr><td>TERMP_01984</td><td>Yes</td><td>1</td><td>Yes</td><td>68.1</td><td>Lipoprotein</td><td></td><td>No</td></tr><tr><td>TERMP_01543</td><td>Yes</td><td>1</td><td>Yes</td><td>45.2</td><td>Uncharacterized protein</td><td>Eight-cysteine-cluster domain</td><td>No</td></tr><tr><td>TERMP_02023</td><td>Yes</td><td>1</td><td>Yes</td><td>22</td><td>Flagellin</td><td></td><td>Yes</td></tr><tr><td>TERMP_00515</td><td>Yes</td><td>0.5</td><td>Yes</td><td>70.5</td><td>S-layer_C domain-containing protein</td><td></td><td>No</td></tr><tr><td>TERMP_01415</td><td>Yes</td><td>0.5</td><td>Yes</td><td>71.6</td><td>Uncharacterized protein</td><td>Hypothetical protein</td><td>No</td></tr><tr><td>TERMP_01455</td><td>Yes</td><td>0.5</td><td>Yes</td><td>81</td><td>Cyclomaltodextrin glucanotransferase</td><td></td><td>No</td></tr><tr><td>TERMP_01372</td><td>Yes</td><td>1</td><td>Yes</td><td>56.4</td><td>Uncharacterized protein</td><td>Amylopullulanase</td><td>No</td></tr><tr><td>TERMP_02026</td><td>Yes</td><td>1</td><td>Yes</td><td>51</td><td>Uncharacterized protein</td><td>Hypothetical protein</td><td>No</td></tr><tr><td>TERMP_00403</td><td>Yes</td><td>1</td><td>No</td><td>8.9</td><td>RNA-binding protein</td><td></td><td>No</td></tr><tr><td>TERMP_01263</td><td>Yes</td><td>1</td><td>No</td><td>27.7</td><td>Metal-dependent hydrolase</td><td></td><td>No</td></tr><tr><td>TERMP_00076</td><td>Yes</td><td>1</td><td>No</td><td>18.8</td><td>Phosphoesterase</td><td></td><td>No</td></tr><tr><td>TERMP_01902</td><td>Yes</td><td>1</td><td>No</td><td>17.6</td><td>Peptidyl-prolyl cis-trans isomerase</td><td></td><td>No</td></tr><tr><td>TERMP_02030</td><td>Yes</td><td>1</td><td>No</td><td>22</td><td>GMP synthase</td><td></td><td>No</td></tr><tr><td>TERMP_02179</td><td>Yes</td><td>1</td><td>Yes</td><td>209.5</td><td>Peptidase_S8 domain-containing protein</td><td></td><td>No</td></tr><tr><td>TERMP_00275</td><td>Yes</td><td>0.5</td><td>No</td><td>86.8</td><td>Maltose phosphorylase</td><td></td><td>No</td></tr><tr><td>TERMP_00281</td><td>Yes</td><td>0.5</td><td>No</td><td>40.1</td><td>Sugar ABC transporter</td><td></td><td>No</td></tr><tr><td>TERMP_01500</td><td>Yes</td><td>1</td><td>No</td><td>8.2</td><td>RNA-binding protein</td><td></td><td>Yes</td></tr><tr><td>TERMP_00360</td><td>No</td><td>1</td><td>No</td><td>21.2</td><td>DNA protection during starvation protein</td><td></td><td>No</td></tr><tr><td>TERMP_00817</td><td>No</td><td>1</td><td>No</td><td>21.5</td><td>Thymidine kinase</td><td></td><td>No</td></tr><tr><td>TERMP_01559</td><td>No</td><td>1</td><td>No</td><td>12</td><td>Ketoisovalerate oxidoreductase subunit</td><td></td><td>No</td></tr><tr><td>TERMP_01449</td><td>Yes</td><td>1</td><td>Yes</td><td>76.4</td><td>Dockerin domain-containing protein</td><td></td><td>No</td></tr><tr><td>TERMP_01791</td><td>No</td><td>1</td><td>Yes</td><td>48.5</td><td>S-layer-like array protein</td><td></td><td>No</td></tr><tr><td>TERMP_00278</td><td>Yes</td><td>0.5</td><td>Yes</td><td>50.9</td><td>MalE-like Mal/Max ABC transporter</td><td></td><td>No</td></tr><tr><td>TERMP_02126</td><td>No</td><td>1</td><td>Yes</td><td>179.8</td><td>vWFA domain-containing protein</td><td></td><td>No</td></tr></tbody><tfoot><tr><td colspan="8" style="text-align: right;">(Continues)</td></tr></tfoot></table>
<table><caption>TABLE 2 (Continued)</caption><thead><tr><th>Ordered locus</th><th>Found only in the supernatant</th><th>Secretion probability</th><th>N-terminal signal sequence<sup>a</sup></th><th>Molecular weight</th><th>Description</th><th>Pfam/UCSC archaeal genome<sup>b</sup></th><th>TrmBL4 binding motif<sup>c</sup></th></tr></thead><tbody><tr><td>TERMP_02130</td><td>Yes</td><td>1</td><td>Yes</td><td>21.8</td><td>Methyltransferase</td><td></td><td>No</td></tr><tr><td>TERMP_00292</td><td>No</td><td>1</td><td>Yes</td><td>54.3</td><td>PEGA domain-containing protein</td><td></td><td>Yes</td></tr><tr><td>TERMP_00597</td><td>No</td><td>1</td><td>Yes</td><td>80.6</td><td>NPCBM_assoc domain-containing protein</td><td>Domain of α-galactosidase</td><td>No</td></tr><tr><td>TERMP_00008</td><td>No</td><td>1</td><td>No</td><td>38.5</td><td>Glycosidase</td><td></td><td>No</td></tr><tr><td>TERMP_01204</td><td>No</td><td>1</td><td>Yes</td><td>22.8</td><td>Flagellin</td><td></td><td>No</td></tr><tr><td>TERMP_01556</td><td>No</td><td>1</td><td>No</td><td>12</td><td>Pyruvate: ferredoxin oxidoreductase Δsubunit</td><td></td><td>No</td></tr><tr><td>TERMP_00750</td><td>No</td><td>1</td><td>Yes</td><td>76.3</td><td>CARDB domain-containing protein</td><td></td><td>No</td></tr><tr><td>TERMP_01657</td><td>No</td><td>1</td><td>Yes</td><td>153.6</td><td>Amylo pullulanase</td><td></td><td>Yes</td></tr></tbody></table>

Note: The supernatant only represents the proteins found only in the supernatant, not in the cytosol; the secretion probability is calculated according to the equations in Data S2 and Mal/Max for maltose/maltodextrin.

<sup>a</sup>Prediction of N-terminal signal sequence using the OutCyte 1.0 program (Zhao et al., 2019).

<sup>b</sup>Significant hit obtained after a search in Pfam 34.0 (Mistry et al., 2021) and the UCSC Archaeal Genome Browser database for hypothetical proteins (Schneider et al., 2006).

<sup>c</sup>Search for the TrmB-like binding motif performed in silico on the whole genome of *T. barophilus*, based on the known TrmB binding motifs in certain Thermococcales.

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>FIGURE 5 Comparison of the relative expression of the genes of interest representative of the target clusters under different conditions in the presence and absence of maltose, showing the effect of *trmBL4* gene mutation (A) and the effect of sugar (B). 1. and 2. *TERMP_00276* and *TERMP_00278* representative of the *TERMP_*(00275–00281) cluster; 3. *TERMP_01652* representative of the *TERMP_*(01652–01658) cluster; 4. *TERMP_01654* representative of *TERMP_*(01652–01658); 5. *TERMP_01835* representative of *TERMP_*(01835–01840).</figcaption></figure>

pos. 1,806,176–1,806,183; and TACTCN<sub>3</sub>CATA, pos. 246,815–246,826) were detected upstream of *TERMP_01500*, *TERMP_02023*, and *TERMP_00292*, respectively. In addition, a complete TGM motif was detected upstream of *TERMP_01654* (MD system) at position 1,441,219–1,441,235. Surprisingly, no TGM sequence was detected upstream of the *TERMP_*(00278–00281) operon or in other regulated glycolytic genes.

DISCUSSION

Little is known about the secretome of *T. barophilus*, which plays an important role in sugar uptake and metabolism. In this study, we made the first analysis of the carbohydrate metabolism in *T. barophilus* by studying its secretome when cultured in the presence of carbohydrates at $P_{atm}$ using genetic, physiological, and
<table><caption>TABLE 2 (Continued)</caption><thead><tr><th>Ordered locus</th><th>Found only in the supernatant</th><th>Secretion probability</th><th>N-terminal signal sequence<sup>a</sup></th><th>Molecular weight</th><th>Description</th><th>Pfam/UCSC archaeal genome<sup>b</sup></th><th>TrmBL4 binding motif<sup>c</sup></th></tr></thead><tbody><tr><td>TERMP_02130</td><td>Yes</td><td>1</td><td>Yes</td><td>21.8</td><td>Methyltransferase</td><td></td><td>No</td></tr><tr><td>TERMP_00292</td><td>No</td><td>1</td><td>Yes</td><td>54.3</td><td>PEGA domain-containing protein</td><td></td><td>Yes</td></tr><tr><td>TERMP_00597</td><td>No</td><td>1</td><td>Yes</td><td>80.6</td><td>NPCBM_assoc domain-containing protein</td><td>Domain of α-galactosidase</td><td>No</td></tr><tr><td>TERMP_00008</td><td>No</td><td>1</td><td>No</td><td>38.5</td><td>Glycosidase</td><td></td><td>No</td></tr><tr><td>TERMP_01204</td><td>No</td><td>1</td><td>Yes</td><td>22.8</td><td>Flagellin</td><td></td><td>No</td></tr><tr><td>TERMP_01556</td><td>No</td><td>1</td><td>No</td><td>12</td><td>Pyruvate: ferredoxin oxidoreductase Δsubunit</td><td></td><td>No</td></tr><tr><td>TERMP_00750</td><td>No</td><td>1</td><td>Yes</td><td>76.3</td><td>CARDB domain-containing protein</td><td></td><td>No</td></tr><tr><td>TERMP_01657</td><td>No</td><td>1</td><td>Yes</td><td>153.6</td><td>Amylo pullulanase</td><td></td><td>Yes</td></tr></tbody></table>

Note: The supernatant only represents the proteins found only in the supernatant, not in the cytosol; the secretion probability is calculated according to the equations in Data S2 and Mal/Max for maltose/maltodextrin.

<sup>a</sup>Prediction of N-terminal signal sequence using the OutCyte 1.0 program (Zhao et al., 2019).

<sup>b</sup>Significant hit obtained after a search in Pfam 34.0 (Mistry et al., 2021) and the UCSC Archaeal Genome Browser database for hypothetical proteins (Schneider et al., 2006).

<sup>c</sup>Search for the TrmB-like binding motif performed in silico on the whole genome of *T. barophilus*, based on the known TrmB binding motifs in certain Thermococcales.

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>FIGURE 5 Comparison of the relative expression of the genes of interest representative of the target clusters under different conditions in the presence and absence of maltose, showing the effect of *trmBL4* gene mutation (A) and the effect of sugar (B). 1. and 2. *TERMP_00276* and *TERMP_00278* representative of the *TERMP_*(00275–00281) cluster; 3. *TERMP_01652* representative of the *TERMP_*(01652–01658) cluster; 4. *TERMP_01654* representative of *TERMP_*(01652–01658); 5. *TERMP_01835* representative of *TERMP_*(01835–01840).</figcaption></figure>

pos. 1,806,176–1,806,183; and TACTCN<sub>3</sub>CATA, pos. 246,815–246,826) were detected upstream of *TERMP_01500*, *TERMP_02023*, and *TERMP_00292*, respectively. In addition, a complete TGM motif was detected upstream of *TERMP_01654* (MD system) at position 1,441,219–1,441,235. Surprisingly, no TGM sequence was detected upstream of the *TERMP_*(00278–00281) operon or in other regulated glycolytic genes.

DISCUSSION

Little is known about the secretome of *T. barophilus*, which plays an important role in sugar uptake and metabolism. In this study, we made the first analysis of the carbohydrate metabolism in *T. barophilus* by studying its secretome when cultured in the presence of carbohydrates at $P_{atm}$ using genetic, physiological, and
<figure><img src="image_19.png" /></figure>

proteomics approaches. Our work represents the first step towards deciphering the organism's mechanism of adaptation to HHP. The regulation of sugar ABC transporters was assessed using deletion mutants of the transcriptional regulator of the TrmB family (TrmBL4) present in *T. barophilus*, which is known to affect the transcription of genes involved in sugar metabolism in Thermococcales. The effect of the deletion of the *trmBL4* gene was reflected by the growth enhancement of the mutant cells compared with the WT in the presence of sugars and these improvements were more pronounced in the absence of sulphur. Regardless of the mutation, the cells in a sulphur-free medium relied more on the sugars to grow and seemed to use sugars as a source of carbon for growth. These results are supported by studies showing that sulphur may play a role in the metabolism of sugars in Archaea (Adams et al., 2001).

The proteomic results highlight critical proteins of the *T. barophilus* secretome, which are probably at least regulated by the *trmBL4* gene product, and others might be impacted by cultivation under non-optimal growth pressure in *T. barophilus*. The STRING database search for protein-protein interactions (Szklarczyk et al., 2019) of the hypothetical amylopullulanase encoded by *TERMP_01372*, repressed in the mutant cells, revealed that the adjacent interacting proteins correspond to a secretion system. This probably indicates a new sugar-related secretion system under the control of the *trmBL4* gene product. Additionally, the interacting proteins encoded by *TERMP_02026* and *TERMP_02023*, repressed in the mutant cells, could have formed a cross-network regulating the cell shape and controlling the timing of the cell division in our tested conditions under the control of the *trmBL4* gene. This is likely because the adjacent interacting proteins, such as a tubulin cell division FtsZ-like protein (*TERMP_02021*), are involved in the control of cell division.

Surprisingly, the protein responsible for pyruvate conversion was expressed under all conditions, with just slight repression in the mutant cells. The presence of this enzyme can be explained either by the use of pyruvate to cope with non-optimal pressure or the activation of the conversion of pyruvate due to its accumulation in the cell. This pyruvate could thus be mobilized under stressful pressure conditions, as in the case of glutamate biosynthesis in *Thermococcus piezophilus* (Moalic et al., 2021).

Additionally, some proteins were found to be over-expressed in the mutant cells. These included a methyltransferase encoded by *TERMP_02130*, which may play a role in controlling the profile of gene expression impacted by the mutation. In addition, the search made in the STRING database (Szklarczyk et al., 2019, 2023) on another overexpressed key protein encoded by *TERMP_00597*, showed that the adjacent

interacting proteins correspond to ABC transporters, which may indicate their disposition in a potential operon involved in sugar transport.

Contrastingly, the expression of the DNA protection during starvation protein and the vWFA domain-containing protein in all tested conditions may indicate a stress response due to the alteration of nutrient uptake under non-optimal pressure conditions (0.1 MPa) and modulation of the cell signalling pathways at 0.1 MPa, respectively.

Both the *trmBL4* gene mutation and the carbohydrates were able, nonetheless, to repress the expression of the protein encoded by *TERMP_01543* as though, in the absence of sugars, the presence of the *trmBL4* gene activates its expression. Therefore, we took a closer look at this protein to decipher its role. *TERMP_01543* may contain an eight-cysteine-cluster domain that could play a role in the biofilm formation as in the fungus *Candida albicans* (Pérez et al. 2006). Indeed, it is known that Archaea biofilms are formed by extracellular polymeric substances composed of polysaccharides, proteins, and metal ions (Stoodley et al., 2002). Studies have indicated that biofilm formation in Archaea provides benefits to the cells, including protection against stressful growth conditions such as toxic chemicals and low or high pH (Koerdt et al., 2010; Megaw & Gilmore, 2017). The phenotypic shifts in the behaviour of Archaea caused by stressful growth conditions require many regulatory processes and lead to the development of biofilms (An & Parsek, 2007). Here, in *T. barophilus*, the mutation of the *trmBL4* gene, addition of sugars, and growth under non-optimal pressure conditions may have altered the biofilm formation by repressing genes exhibiting an enhanced ability to form biofilms. In *S. solfataricus*, the products of β-galactosidase and α-mannosidase can control the saccharide volume in the biofilm matrix (Koerdt et al., 2010).

Based on the proteomic results and the KEGG pathway database (Kanehisa, 2002), it is clear that maltose and maltodextrin share some enzymes and thus some common pathways, but pectin may not share the main assimilation systems with maltose and maltodextrin due to its complexity. Pectin is known to be degraded by specific pectinolytic enzymes with the corresponding transporters, as in the pectin degradation pathway in *Erwinia chrysanthemi* (now reclassified as *Dickeya dadantii*; Hugouvieux-Cotte-Pattat et al., 1996).

The S-layer domain protein encoded by *TERMP_01807*, which was only overexpressed in the presence of maltodextrin in the WT cells, is probably involved in a specific maltodextrin membrane transport system because its adjacent interacted protein encoded by *TERMP_01808* corresponds to a major facilitator superfamily transporter. Additionally, we think that the expression of proteins forming part of the S-layer domain is also pressure-dependent, highli
proteomics approaches. Our work represents the first step towards deciphering the organism's mechanism of adaptation to HHP. The regulation of sugar ABC transporters was assessed using deletion mutants of the transcriptional regulator of the TrmB family (TrmBL4) present in *T. barophilus*, which is known to affect the transcription of genes involved in sugar metabolism in Thermococcales. The effect of the deletion of the *trmBL4* gene was reflected by the growth enhancement of the mutant cells compared with the WT in the presence of sugars and these improvements were more pronounced in the absence of sulphur. Regardless of the mutation, the cells in a sulphur-free medium relied more on the sugars to grow and seemed to use sugars as a source of carbon for growth. These results are supported by studies showing that sulphur may play a role in the metabolism of sugars in Archaea (Adams et al., 2001).

The proteomic results highlight critical proteins of the *T. barophilus* secretome, which are probably at least regulated by the *trmBL4* gene product, and others might be impacted by cultivation under non-optimal growth pressure in *T. barophilus*. The STRING database search for protein-protein interactions (Szklarczyk et al., 2019) of the hypothetical amylopullulanase encoded by *TERMP_01372*, repressed in the mutant cells, revealed that the adjacent interacting proteins correspond to a secretion system. This probably indicates a new sugar-related secretion system under the control of the *trmBL4* gene product. Additionally, the interacting proteins encoded by *TERMP_02026* and *TERMP_02023*, repressed in the mutant cells, could have formed a cross-network regulating the cell shape and controlling the timing of the cell division in our tested conditions under the control of the *trmBL4* gene. This is likely because the adjacent interacting proteins, such as a tubulin cell division FtsZ-like protein (*TERMP_02021*), are involved in the control of cell division.

Surprisingly, the protein responsible for pyruvate conversion was expressed under all conditions, with just slight repression in the mutant cells. The presence of this enzyme can be explained either by the use of pyruvate to cope with non-optimal pressure or the activation of the conversion of pyruvate due to its accumulation in the cell. This pyruvate could thus be mobilized under stressful pressure conditions, as in the case of glutamate biosynthesis in *Thermococcus piezophilus* (Moalic et al., 2021).

Additionally, some proteins were found to be over-expressed in the mutant cells. These included a methyltransferase encoded by *TERMP_02130*, which may play a role in controlling the profile of gene expression impacted by the mutation. In addition, the search made in the STRING database (Szklarczyk et al., 2019, 2023) on another overexpressed key protein encoded by *TERMP_00597*, showed that the adjacent

interacting proteins correspond to ABC transporters, which may indicate their disposition in a potential operon involved in sugar transport.

Contrastingly, the expression of the DNA protection during starvation protein and the vWFA domain-containing protein in all tested conditions may indicate a stress response due to the alteration of nutrient uptake under non-optimal pressure conditions (0.1 MPa) and modulation of the cell signalling pathways at 0.1 MPa, respectively.

Both the *trmBL4* gene mutation and the carbohydrates were able, nonetheless, to repress the expression of the protein encoded by *TERMP_01543* as though, in the absence of sugars, the presence of the *trmBL4* gene activates its expression. Therefore, we took a closer look at this protein to decipher its role. *TERMP_01543* may contain an eight-cysteine-cluster domain that could play a role in the biofilm formation as in the fungus *Candida albicans* (Pérez et al. 2006). Indeed, it is known that Archaea biofilms are formed by extracellular polymeric substances composed of polysaccharides, proteins, and metal ions (Stoodley et al., 2002). Studies have indicated that biofilm formation in Archaea provides benefits to the cells, including protection against stressful growth conditions such as toxic chemicals and low or high pH (Koerdt et al., 2010; Megaw & Gilmore, 2017). The phenotypic shifts in the behaviour of Archaea caused by stressful growth conditions require many regulatory processes and lead to the development of biofilms (An & Parsek, 2007). Here, in *T. barophilus*, the mutation of the *trmBL4* gene, addition of sugars, and growth under non-optimal pressure conditions may have altered the biofilm formation by repressing genes exhibiting an enhanced ability to form biofilms. In *S. solfataricus*, the products of β-galactosidase and α-mannosidase can control the saccharide volume in the biofilm matrix (Koerdt et al., 2010).

Based on the proteomic results and the KEGG pathway database (Kanehisa, 2002), it is clear that maltose and maltodextrin share some enzymes and thus some common pathways, but pectin may not share the main assimilation systems with maltose and maltodextrin due to its complexity. Pectin is known to be degraded by specific pectinolytic enzymes with the corresponding transporters, as in the pectin degradation pathway in *Erwinia chrysanthemi* (now reclassified as *Dickeya dadantii*; Hugouvieux-Cotte-Pattat et al., 1996).

The S-layer domain protein encoded by *TERMP_01807*, which was only overexpressed in the presence of maltodextrin in the WT cells, is probably involved in a specific maltodextrin membrane transport system because its adjacent interacted protein encoded by *TERMP_01808* corresponds to a major facilitator superfamily transporter. Additionally, we think that the expression of proteins forming part of the S-layer domain is also pressure-dependent, highli
<figure><img src="image_20.png" /></figure>

ghting their role in cell stabilization under sub-optimal pressure (0.1 MPa) since *TERMP_01807* was over-expressed at 0.1 versus 40 MPa (Vannier et al., 2015). This phenomenon was also observed in the gene expression patterns of *T. piezophilus* (Moalic et al., 2021).

Intriguingly, the expression of RNA-binding proteins requires the addition of sugars in the mutant cells, but not the WT cells. In other words, the presence of the *trmBL4* gene compensates for the absence of sugar. These proteins seem to have a role in regulating gene expression in our different tested conditions (Murina & Nikulin, 2011).

The presence of proteins in the supernatant of the cells devoid of a signal peptide can be explained by cell lysis, the limit of detection of the OutCyte program, or by them being classified as moonlighting proteins (Jeffery, 2003; Huberts & Van der Klei, 2010), which are uni-functional proteins able to operate on both intracellular and extracellular levels (Sengupta et al., 2008). Particularly in the case of Archaea, another possible explanation is the export of proteins via membrane vesicles (Ellen et al., 2009; Ellen et al., 2010; Soler et al., 2008). More work is needed to determine which of these possible explanations are valid.

Interestingly, the RT-qPCR results highlight a particular regulatory mechanism of the *trmBL4* gene product on some HHP-responsive gene clusters involved in carbohydrate metabolism. In the absence of maltose, TrmBL4 seems to activate gene expression but, in the presence of this sugar, it acts as a repressor of gene expression. In our case, sugars did not rescue transcription as seen in *Pfu-TrmBL1* (Lee et al., 2008; Lee, Surma, et al., 2007). So, in the presence of maltose, the *trmBL4* regulator seems to act as a global transcriptional repressor of the *TERMP_(00275-00281)* cluster, with high binding affinity, and of the *TERMP_(01652-01658)* cluster (MD system), with lower affinity, defining it as a regulon. It should also be mentioned that the regulation of the expression of genes is not directly related to the composition of the secretome. The absence of TrmBL4 can have many more repercussions than those observed on the target genes tested by RT-qPCR, such as effects on other target genes, retro-control, and protein-protein interactions, which could all consecutively modify the secretome of *T. barophilus*.

In particular, the LC-MS/MS and RT-qPCR results may indicate synergy between TrmBL4 and TrmBL1, since each of these may regulate the MD operon but with different affinities. Indeed, the expression of the *trmBL1* gene encoded by *TERMP_01652* is increased in the mutant, as shown by the LC/MS-MS (Data S1) and the RT-qPCR results, as though compensating for the mutation of one of the regulators, the *trmBL4* gene. Consequently, we propose that these two members of the TrmB family are involved in a cross-regulatory network of sugar-sensing transcriptional regulators, as in

the case of *P. furiosus*, characterized by the multiplicity of regulators acting on the MD promoter (Lee et al., 2008). Thus, in the absence of maltose, the TrmBL4 regulator alone may repress its expression leading to the de-repression of the targeted genes, but repression is otherwise activated by maltose. This would mean that maltose stimulates the synthesis of TrmBL4, which would in turn repress the expression of genes encoding glycolytic enzymes. This emphasizes the important role of sugars in controlling transport and glycolysis. Moreover, TrmBL4 had no detectable effect on the *TERMP_(01835-01840)* operon. Interestingly, this operon was previously found to be overexpressed at high pressure (40 MPa; Vannier et al., 2015) but was not detected at 0.1 MPa.

The *in-silico* screening for TrmBL4 binding sites indicates a certain degree of conservation, showing a divergence linked to the specific evolution of TrmBL4 compared with other TrmB-like regulators. The latter need to be studied by footprint analysis on the promoter regions. The TrmBL4 target binding site also does not seem to be restricted to a TGM sequence because it is also involved in the regulation of genes with non-TGM-containing sequences.

## CONCLUSION

This study presents the first description of sugar metabolism gene products in *T. barophilus* and the potential regulation of some of these by the *trmBL4* gene product at atmospheric pressure. Many valuable proteins have been identified that could be the subject of further molecular studies in the near future. Some of the gene clusters coding for these proteins appear to be under the control of TrmBL4, one of the five TrmB-like transcriptional regulators in the *T. barophilus* genome. It would be interesting to compare the composition of the secretome in the presence of sugars according to the presence or not of sulphur. It seems that, in a free sulphur medium, the sugars participate more actively in the energy metabolism and should therefore be more assimilated by the cells. Also, the regulation of the expression of the genes by TrmBL4 described in this study needs to be carried out at HHP because some genes of sugar metabolism are known to be responsive to these conditions. This is the first study of a TrmB family regulator in *T. barophilus*, opening up the possibility of a subsequent in-depth study to characterize the gene expression regulation mechanisms of sugar metabolism structurally, molecularly, genetically, and biochemically in relation to hydrostatic pressure.

## AUTHOR CONTRIBUTIONS

**Maria Batour:** Methodology (equal); writing—original draft (equal). **Sebastien Laurent:** Data curation (equal); methodology (equal); writing—review and
<figure><img src="image_18.png" /></figure>

ghting their role in cell stabilization under sub-optimal pressure (0.1 MPa) since *TERMP_01807* was over-expressed at 0.1 versus 40 MPa (Vannier et al., 2015). This phenomenon was also observed in the gene expression patterns of *T. piezophilus* (Moalic et al., 2021).

Intriguingly, the expression of RNA-binding proteins requires the addition of sugars in the mutant cells, but not the WT cells. In other words, the presence of the *trmBL4* gene compensates for the absence of sugar. These proteins seem to have a role in regulating gene expression in our different tested conditions (Murina & Nikulin, 2011).

The presence of proteins in the supernatant of the cells devoid of a signal peptide can be explained by cell lysis, the limit of detection of the OutCyte program, or by them being classified as moonlighting proteins (Jeffery, 2003; Huberts & Van der Klei, 2010), which are uni-functional proteins able to operate on both intracellular and extracellular levels (Sengupta et al., 2008). Particularly in the case of Archaea, another possible explanation is the export of proteins via membrane vesicles (Ellen et al., 2009; Ellen et al., 2010; Soler et al., 2008). More work is needed to determine which of these possible explanations are valid.

Interestingly, the RT-qPCR results highlight a particular regulatory mechanism of the *trmBL4* gene product on some HHP-responsive gene clusters involved in carbohydrate metabolism. In the absence of maltose, TrmBL4 seems to activate gene expression but, in the presence of this sugar, it acts as a repressor of gene expression. In our case, sugars did not rescue transcription as seen in *Pfu-TrmBL1* (Lee et al., 2008; Lee, Surma, et al., 2007). So, in the presence of maltose, the *trmBL4* regulator seems to act as a global transcriptional repressor of the *TERMP_(00275-00281)* cluster, with high binding affinity, and of the *TERMP_(01652-01658)* cluster (MD system), with lower affinity, defining it as a regulon. It should also be mentioned that the regulation of the expression of genes is not directly related to the composition of the secretome. The absence of TrmBL4 can have many more repercussions than those observed on the target genes tested by RT-qPCR, such as effects on other target genes, retro-control, and protein-protein interactions, which could all consecutively modify the secretome of *T. barophilus*.

In particular, the LC-MS/MS and RT-qPCR results may indicate synergy between TrmBL4 and TrmBL1, since each of these may regulate the MD operon but with different affinities. Indeed, the expression of the *trmBL1* gene encoded by *TERMP_01652* is increased in the mutant, as shown by the LC/MS-MS (Data S1) and the RT-qPCR results, as though compensating for the mutation of one of the regulators, the *trmBL4* gene. Consequently, we propose that these two members of the TrmB family are involved in a cross-regulatory network of sugar-sensing transcriptional regulators, as in

the case of *P. furiosus*, characterized by the multiplicity of regulators acting on the MD promoter (Lee et al., 2008). Thus, in the absence of maltose, the TrmBL4 regulator alone may repress its expression leading to the de-repression of the targeted genes, but repression is otherwise activated by maltose. This would mean that maltose stimulates the synthesis of TrmBL4, which would in turn repress the expression of genes encoding glycolytic enzymes. This emphasizes the important role of sugars in controlling transport and glycolysis. Moreover, TrmBL4 had no detectable effect on the *TERMP_(01835-01840)* operon. Interestingly, this operon was previously found to be overexpressed at high pressure (40 MPa; Vannier et al., 2015) but was not detected at 0.1 MPa.

The *in-silico* screening for TrmBL4 binding sites indicates a certain degree of conservation, showing a divergence linked to the specific evolution of TrmBL4 compared with other TrmB-like regulators. The latter need to be studied by footprint analysis on the promoter regions. The TrmBL4 target binding site also does not seem to be restricted to a TGM sequence because it is also involved in the regulation of genes with non-TGM-containing sequences.

## CONCLUSION

This study presents the first description of sugar metabolism gene products in *T. barophilus* and the potential regulation of some of these by the *trmBL4* gene product at atmospheric pressure. Many valuable proteins have been identified that could be the subject of further molecular studies in the near future. Some of the gene clusters coding for these proteins appear to be under the control of TrmBL4, one of the five TrmB-like transcriptional regulators in the *T. barophilus* genome. It would be interesting to compare the composition of the secretome in the presence of sugars according to the presence or not of sulphur. It seems that, in a free sulphur medium, the sugars participate more actively in the energy metabolism and should therefore be more assimilated by the cells. Also, the regulation of the expression of the genes by TrmBL4 described in this study needs to be carried out at HHP because some genes of sugar metabolism are known to be responsive to these conditions. This is the first study of a TrmB family regulator in *T. barophilus*, opening up the possibility of a subsequent in-depth study to characterize the gene expression regulation mechanisms of sugar metabolism structurally, molecularly, genetically, and biochemically in relation to hydrostatic pressure.

## AUTHOR CONTRIBUTIONS

**Maria Batour:** Methodology (equal); writing—original draft (equal). **Sebastien Laurent:** Data curation (equal); methodology (equal); writing—review and
<figure><img src="image_21.png" /></figure>

editing (equal). **Yann Moalic:** Formal analysis (equal); methodology (equal); writing-review and editing (equal). **Hala Chamieh:** Supervision (supporting); writing-review and editing (equal). **Samir Taha:** Writing-review and editing (supporting). **Mohamed Jebbar:** Conceptualization (lead); funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); validation (lead); writing—original draft (lead); writing—review and editing (lead).

## ACKNOWLEDGEMENTS

We thank Nadège Quintin for providing the strains used in this work, which are part of the UBO culture collection. We are indebted to C. Henry of the Paris Sud Ouest PAPPSO proteomics core facility (supported by INRA), the Ile-de-France regional council, IBiSA, and the CNRS for the LC/MS analyses. We are grateful to Helen McCombie of the University Translation Office for improving the language. This work was conducted by Maria Batour as part of her PhD work co-supervised by the University of Lebanon and the University of Brest. Maria Batour was supported by a PhD fellowship from the Lebanese University and the municipality of Kadaa Zgharta. Maria Batour also received support from the IsBlue, an Interdisciplinary Graduate School for the Blue Planet (ANR-17-EURE-0015) and co-funded by a grant from the French government under the program ‘Investissements d'Avenir', which is part of the France 2030 plan. Yann Moalic was supported by a postdoctoral fellowship from the French Agence Nationale de la Recherche (ANR-16-CE12-0016). Financial support was provided by the CNRS, Univ Brest, Ifremer, IsBlue, and the Lebanese University.

## CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

## DATA AVAILABILITY STATEMENT

Datasets can be found in the Supplementary Data files.

## ORCID

Yann Moalic https://orcid.org/0000-0001-7385-7484

Mohamed Jebbar https://orcid.org/0000-0003-3879-4400

## REFERENCES

Adams, M.W., Holden, J.F., Menon, A.L., Schut, G.J., Grunden, A.M., Hou, C. et al. (2001) Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon *Pyrococcus furiosus*. *Journal of Bacteriology*, 183(2), 716–724.

An, D. & Parsek, M.R. (2007) The promise and peril of transcriptional profiling in biofilm communities. *Current Opinion in Microbiology*, 10, 292–296.

Bertoldo, C. & Antranikian, G. (2006) The order Thermococcales. In: Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.H. & Stackebrandt, E. (Eds.) *The prokaryotes*. New York, NY: Springer.

Birien, T., Thiel, A., Henneke, G., Flament, D., Moalic, Y. & Jebbar, M. (2018) Development of an effective 6-methylpurine counterselection marker for genetic manipulation in *Thermococcus barophilus*. *Genes*, 9(2), 77.

Blamey, J.M. & Adams, M.W. (1993) Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon *Pyrococcus furiosus*. *Biochimica et Biophysica Acta*, 1161(1), 19–27.

Constantino, H.R., Brown, S.H. & Kelly, R.M. (1990) Purification and characterization of an α-glucosidase from a hyperthermophilic archebacterium, *Pyrococcus furiosus*, exhibiting a temperature optimum of 105°C and 115°C. *Journal of Bacteriology*, 172, 3654–3660.

Durbin, R., Eddy, S.R., Krogh, A. & Mitchison, G. (1998) *Biological sequence analysis: probabilistic models of proteins and nucleic acids*. Cambridge, UK: Cambridge University Press.

Ellen, A.F., Albers, S.V. & Driessen, A.J.M. (2010) Comparative study of the extracellular proteome of *Sulfolobus* species reveals limited secretion. *Extremophiles*, 14, 87–98.

Ellen, A.F., Albers, S.V., Huibers, W., Pitcher, A., Hobel, C.F., Schwarz, H. et al. (2009) Proteomic analysis of secreted membrane vesicles of archaeal *Sulfolobus* species reveals the presence of endosome sorting complex components. *Extremophiles*, 13, 67–79.

Frenkiel-Krispin, D., Levin-Zaidman, S., Shimoni, E., Wolf, S.G., Wachtel, E.J., Arad, T. et al. (2001) Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection. *The EMBO Journal*, 20(5), 1184–1191.

Gindner, A., Hausner, W. & Thomm, M. (2014) The TrmB family: a versatile group of transcriptional regulators in archaea. *Extremophiles*, 18(5), 925–936.

Huberts, D.H. & Van der Klei, I.J. (2010) Moonlighting proteins: an intriguing mode of multitasking. *Biochimica et Biophysica Acta*, 1803, 520–525.

Hugouvieux-Cotte-Pattat, N., Condemine, G., Nasser, W. & Reverchon, S. (1996) Regulation of pectinolysis in *Erwinia chrysanthemi*. *Annual Review of Microbiology*, 50, 213–257.

Jeffery, C.J. (2003) Moonlighting proteins: old proteins learning new tricks. *Trends in Genetics*, 19(8), 415–417.

Kanehisa, M. (2002) The KEGG database. *Novartis Foundation Symposium*, 247, 91–252.

Kengen, S.W.M., Luesink, E.J., Stams, A.J. & Zehnder, A.J. (1993) Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon *Pyrococcus furiosus*. *The Journal of Biological Chemistry*, 213, 305–312.

Kim, M., Park, S. & Lee, S.J. (2016) Global transcriptional regulator TrmB family members in prokaryotes. *Journal of Microbiology*, 54, 639–645.

Koerdt, A., Gödeke, J., Berger, J., Thormann, K.M. & Albers, S.V. (2010) Crenarchaeal biofilm formation under extreme conditions. *PLoS One*, 5(11), e14104.

Lee, S.J., Engelmann, A., Horlacher, R., Qu, Q., Vierke, G., Hebbeln, C. et al. (2003) TrmB, a sugar-specific transcriptional regulator of the trehalose/maltose ABC transporter from the hyperthermophilic archaeon *Thermococcus litoralis*. *The Journal of Biological Chemistry*, 278(2), 983–990.

Lee, S.J., Moulakakis, C., Koning, S.M., Hausner, W., Thomm, M. & Boos, W. (2005) trmB, a sugar sensing regulator for ABC transporter genes in *Pyrococcus furiosus* exhibits dual promoter specificity and is controlled by different inducers. *Molecular Microbiology*, 57, 1797–1807.

Lee, S.J., Seitz, S., Surma, M., Hausner, W., Thomm, M. & Boos, W. (2007) Differential signal transduction via TrmB, a sugar sensing transcriptional repressor of *Pyrococcus furiosus*. *Molecular Microbiology*, 64(6), 1499–1505.

Lee, S.J., Surma, M., Hausner, W., Thomm, M. & Boos, W. (2008) The role of TrmB and trmBL-like transcriptional regulators for
<figure><img src="image_19.png" /></figure>

editing (equal). **Yann Moalic:** Formal analysis (equal); methodology (equal); writing—review and editing (equal). **Hala Chamieh:** Supervision (supporting); writing—review and editing (equal). **Samir Taha:** Writing—review and editing (supporting). **Mohamed Jebbar:** Conceptualization (lead); funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); validation (lead); writing—original draft (lead); writing—review and editing (lead).

## ACKNOWLEDGEMENTS

We thank Nadège Quintin for providing the strains used in this work, which are part of the UBO culture collection. We are indebted to C. Henry of the Paris Sud Ouest PAPPSO proteomics core facility (supported by INRA), the Ile-de-France regional council, IBiSA, and the CNRS for the LC/MS analyses. We are grateful to Helen McCombie of the University Translation Office for improving the language. This work was conducted by Maria Batour as part of her PhD work co-supervised by the University of Lebanon and the University of Brest. Maria Batour was supported by a PhD fellowship from the Lebanese University and the municipality of Kadaa Zgharta. Maria Batour also received support from the IsBlue, an Interdisciplinary Graduate School for the Blue Planet (ANR-17-EURE-0015) and co-funded by a grant from the French government under the program ‘Investissements d'Avenir', which is part of the France 2030 plan. Yann Moalic was supported by a postdoctoral fellowship from the French Agence Nationale de la Recherche (ANR-16-CE12-0016). Financial support was provided by the CNRS, Univ Brest, Ifremer, IsBlue, and the Lebanese University.

## CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

## DATA AVAILABILITY STATEMENT

Datasets can be found in the Supplementary Data files.

## ORCID

Yann Moalic https://orcid.org/0000-0001-7385-7484

Mohamed Jebbar https://orcid.org/0000-0003-3879-4400

## REFERENCES

Adams, M.W., Holden, J.F., Menon, A.L., Schut, G.J., Grunden, A.M., Hou, C. et al. (2001) Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon *Pyrococcus furiosus*. *Journal of Bacteriology*, 183(2), 716–724.

An, D. & Parsek, M.R. (2007) The promise and peril of transcriptional profiling in biofilm communities. *Current Opinion in Microbiology*, 10, 292–296.

Bertoldo, C. & Antranikian, G. (2006) The order Thermococcales. In: Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.H. & Stackebrandt, E. (Eds.) *The prokaryotes*. New York, NY: Springer.

Birien, T., Thiel, A., Henneke, G., Flament, D., Moalic, Y. & Jebbar, M. (2018) Development of an effective 6-methylpurine counterselection marker for genetic manipulation in *Thermococcus barophilus*. *Genes*, 9(2), 77.

Blamey, J.M. & Adams, M.W. (1993) Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon *Pyrococcus furiosus*. *Biochimica et Biophysica Acta*, 1161(1), 19–27.

Constantino, H.R., Brown, S.H. & Kelly, R.M. (1990) Purification and characterization of an α-glucosidase from a hyperthermophilic archebacterium, *Pyrococcus furiosus*, exhibiting a temperature optimum of 105°C and 115°C. *Journal of Bacteriology*, 172, 3654–3660.

Durbin, R., Eddy, S.R., Krogh, A. & Mitchison, G. (1998) *Biological sequence analysis: probabilistic models of proteins and nucleic acids*. Cambridge, UK: Cambridge University Press.

Ellen, A.F., Albers, S.V. & Driessen, A.J.M. (2010) Comparative study of the extracellular proteome of *Sulfolobus* species reveals limited secretion. *Extremophiles*, 14, 87–98.

Ellen, A.F., Albers, S.V., Huibers, W., Pitcher, A., Hobel, C.F., Schwarz, H. et al. (2009) Proteomic analysis of secreted membrane vesicles of archaeal *Sulfolobus* species reveals the presence of endosome sorting complex components. *Extremophiles*, 13, 67–79.

Frenkiel-Krispin, D., Levin-Zaidman, S., Shimoni, E., Wolf, S.G., Wachtel, E.J., Arad, T. et al. (2001) Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection. *The EMBO Journal*, 20(5), 1184–1191.

Gindner, A., Hausner, W. & Thomm, M. (2014) The TrmB family: a versatile group of transcriptional regulators in archaea. *Extremophiles*, 18(5), 925–936.

Huberts, D.H. & Van der Klei, I.J. (2010) Moonlighting proteins: an intriguing mode of multitasking. *Biochimica et Biophysica Acta*, 1803, 520–525.

Hugouvieux-Cotte-Pattat, N., Condemine, G., Nasser, W. & Reverchon, S. (1996) Regulation of pectinolysis in *Erwinia chrysanthemi*. *Annual Review of Microbiology*, 50, 213–257.

Jeffery, C.J. (2003) Moonlighting proteins: old proteins learning new tricks. *Trends in Genetics*, 19(8), 415–417.

Kanehisa, M. (2002) The KEGG database. *Novartis Foundation Symposium*, 247, 91–252.

Kengen, S.W.M., Luesink, E.J., Stams, A.J. & Zehnder, A.J. (1993) Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon *Pyrococcus furiosus*. *The Journal of Biological Chemistry*, 213, 305–312.

Kim, M., Park, S. & Lee, S.J. (2016) Global transcriptional regulator TrmB family members in prokaryotes. *Journal of Microbiology*, 54, 639–645.

Koerdt, A., Gödeke, J., Berger, J., Thormann, K.M. & Albers, S.V. (2010) Crenarchaeal biofilm formation under extreme conditions. *PLoS One*, 5(11), e14104.

Lee, S.J., Engelmann, A., Horlacher, R., Qu, Q., Vierke, G., Hebbeln, C. et al. (2003) TrmB, a sugar-specific transcriptional regulator of the trehalose/maltose ABC transporter from the hyperthermophilic archaeon *Thermococcus litoralis*. *The Journal of Biological Chemistry*, 278(2), 983–990.

Lee, S.J., Moulakakis, C., Koning, S.M., Hausner, W., Thomm, M. & Boos, W. (2005) trmB, a sugar sensing regulator for ABC transporter genes in *Pyrococcus furiosus* exhibits dual promoter specificity and is controlled by different inducers. *Molecular Microbiology*, 57, 1797–1807.

Lee, S.J., Seitz, S., Surma, M., Hausner, W., Thomm, M. & Boos, W. (2007) Differential signal transduction via TrmB, a sugar sensing transcriptional repressor of *Pyrococcus furiosus*. *Molecular Microbiology*, 64(6), 1499–1505.

Lee, S.J., Surma, M., Hausner, W., Thomm, M. & Boos, W. (2008) The role of TrmB and trmBL-like transcriptional regulators for
<figure><img src="image_22.png" /></figure>

sugar transport and metabolism in the hyperthermophilic
archaeon *Pyrococcus furiosus*. *Archives of Microbiology*, 190(3),
247–256.

Lee, S.J., Surma, M., Seitz, S., Hausner, W., Thomm, M. & Boos, W. (2007) Characterization of the TrmB-like protein, PF0124, a TGM-recognizing global transcriptional regulator of the hyper-thermophilic archaeon *Pyrococcus furiosus*. *Molecular Microbiology*, 65(2), 305–318.

Livak, J.K. & Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 (ΔDelta Delta C(T)) method. *Methods*, 25(4), 402–408.

Marteinsson, V.T., Birrien, J.L. & Prieur, D. (1997) In situ enrichment and isolation of thermophilic microorganisms from deep-sea vent environments. *Canadian Journal of Microbiology*, 43, 694e7–694e697.

Marteinsson, V.T., Birrien, J.L., Reysenbach, A.L., Vernet, M., Marie, D., Gambacorta, A. et al. (1999) *Thermococcus barophilus* sp. nov., a new barophilic and hyperthermophilic archaeon isolated under high hydrostatic pressure from a deep-sea hydrothermal vent. *International Journal of Bacteriology*, 49(2), 351–359.

Maruyama, H., Shin, M., Oda, T., Matsumi, R., Ohniwa, R.L., Itoh, T. et al. (2011) Histone and TK0471/TrmBL2 form a novel hetero-geneous genome architecture in the hyperthermophilic archaeon *Thermococcus kodakarensis*. *Molecular Biology of the Cell*, 22(3), 386–398.

Megaw, J. & Gilmore, B.F. (2017) Archaeal persisters: persister cell formation as a stress response in *Haloferax volcanii*. *Frontiers in Microbiology*, 8, 1589.

Mistry, J., Chuguransky, S., Williams, L., Qureshi, M., Salazar, G.A., Sonnhammer, E. et al. (2021) Pfam: the protein families database in 2021. *Nucleic Acids Research*, 49(D1), D412–D419.

Moalic, Y., Hartunians, J., Dalmasso, C., Courtine, D., Georges, M., Oger, P. et al. (2021) The piezo-hyperthermophilic archaeon *Thermococcus piezophilus* regulates its energy efficiency system to cope with large hydrostatic pressure variations. *Frontiers in Microbiology*, 12, 730231.

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Pérez, A., Pedrós, B., Murgui, A., Casanova, M., López-Ribot, J.L., & Martínez, J.P. (2006) Biofilm formation by Candida albicans mutants for genes coding fungal proteins exhibiting the eight-cysteine-containing CFEM domain. *FEMS Yeast Res*, 6 (7), 1074–1084.

Schmid, G., Mathiesen, G., Arntzen, M.O., Eijsink, V.G. & Thomm, M. (2013) Experimental and computational analysis of the secretome of the hyperthermophilic archaeon *Pyrococcus furiosus*. *Extremophiles*, 17(6), 921–930.

Schmittgen, T.D. & Zakrajsek, B.A. (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. *Journal of Biochemical and Biophysical Methods*, 46(1–2), 69–81.

Schneider, K.L., Pollard, K.S., Baertsch, R., Pohl, A. & Lowe, T.M. (2006) The UCSC archaeal genome browser. *Nucleic Acids Research*, 34 part 2 (Database issue), D407–D410.

Sengupta, S., Ghosh, S. & Nagaraja, V. (2008) Moonlighting function of glutamate racemase from *mycobacterium tuberculosis*: race-mization and DNA gyrase inhibition are two independent activities of the enzyme. *Microbiol*, 154(9), 2796–2803.

Soler, N., Marguet, E., Verbavatz, J.M. & Forterre, P. (2008) Virus-like vesicles and extracellular DNA produced by hyperthermophilic archaea of the order Thermococcales. *Research in Microbiology*, 159, 390–399.

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Szklarczyk, D., Kirsch, R., Koutrouli, M., Nastou, K., Mehryary, F., Hachilif, R. et al. (2023) The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. *Nucleic Acids Research*, 51(D1), D638–D646.

Thiel, A., Michoud, G., Moalic, Y., Flament, D. & Jebbar, M. (2014) Genetic manipulations of the hyperthermophilic piezophilic archaeon *Thermococcus barophilus*. *Applied and Environmental Microbiology*, 80(7), 2299–2306.

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Vannier, P., Marteinsson, V.T., Fridjonsson, O.H., Oger, P. & Jebbar, M. (2011) Complete genome sequence of the hyperthermophilic, piezophilic, heterotrophic, and carboxydotrophic archaeon *Thermococcus barophilus* MP. *Journal of Bacteriology*, 193(6), 1481–1482.

Vannier, P., Michoud, G., Oger, P., Marteinsson, V.P. & Jebbar, M. (2015) Genome expression of *Thermococcus barophilus* and *Thermococcus kodakarensis* in response to different hydrostatic pressure conditions. *Microbiological Research*, 166(9), 717–725.

Waskom, M.L. (2021) seaborn: statistical data visualization. *Journal of Open Source Software*, 6(60), 3021.

Whittaker, C.A. & Hynes, R.O. (2002) Distribution and evolution of von Willebrand/integrin domains: widely dispersed domains with roles in cell adhesion and elsewhere. *Molecular Biology of the Cell*, 13(10), 3369–3387.

Zeng, X., Birrien, J.L., Fouquet, Y., Cherkashov, G., Jebbar, M., Querellou, J. et al. (2009) *Pyrococcus* CH1, an obligate piezophilic hyperthermophile: extending the upper pressure-temperature limits for life. *The ISME Journal*, 3(7), 873–876.

Zhao, L., Poschmann, G., Waldera-Lupa, D., Rafiee, N., Kollmann, M. & Stühler, K. (2019) OutCyte: a novel tool for predicting unconventional protein secretion. *Scientific Reports*, 9(1), 19448.

SUPPORTING INFORMATION

Additional supporting information can be found online
in the Supporting Information section at the end of this
article.

How to cite this article: Batour, M., Laurent, S., Moalic, Y., Chamieh, H., Taha, S. & Jebbar, M. (2023) The secretome of *Thermococcus barophilus* in the presence of carbohydrates and the potential role of the TrmBL4 regulator. *Environmental Microbiology Reports*, 15(6), 530–544. Available from: https://doi.org/10.1111/1758-2229.13186
<figure><img src="image_20.png" /></figure>

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Moalic, Y., Hartunians, J., Dalmasso, C., Courtine, D., Georges, M., Oger, P. et al. (2021) The piezo-hyperthermophilic archaeon *Thermococcus piezophilus* regulates its energy efficiency system to cope with large hydrostatic pressure variations. *Frontiers in Microbiology*, 12, 730231.

Murina, V.N. & Nikulin, A.D. (2011) RNA-binding Sm-like proteins of bacteria and archaea. Similarity and difference in structure and function. *The Biochemist*, 76(13), 1434–1449.

Pérez, A., Pedrós, B., Murgui, A., Casanova, M., López-Ribot, J.L., & Martínez, J.P. (2006) Biofilm formation by Candida albicans mutants for genes coding fungal proteins exhibiting the eight-cysteine-containing CFEM domain. *FEMS Yeast Res*, 6 (7), 1074–1084.

Schmid, G., Mathiesen, G., Arntzen, M.O., Eijsink, V.G. & Thomm, M. (2013) Experimental and computational analysis of the secretome of the hyperthermophilic archaeon *Pyrococcus furiosus*. *Extremophiles*, 17(6), 921–930.

Schmittgen, T.D. & Zakrajsek, B.A. (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. *Journal of Biochemical and Biophysical Methods*, 46(1–2), 69–81.

Schneider, K.L., Pollard, K.S., Baertsch, R., Pohl, A. & Lowe, T.M. (2006) The UCSC archaeal genome browser. *Nucleic Acids Research*, 34 part 2 (Database issue), D407–D410.

Sengupta, S., Ghosh, S. & Nagaraja, V. (2008) Moonlighting function of glutamate racemase from *mycobacterium tuberculosis*: race-mization and DNA gyrase inhibition are two independent activities of the enzyme. *Microbiol*, 154(9), 2796–2803.

Soler, N., Marguet, E., Verbavatz, J.M. & Forterre, P. (2008) Virus-like vesicles and extracellular DNA produced by hyperthermophilic archaea of the order Thermococcales. *Research in Microbiology*, 159, 390–399.

Stoodley, P., Sauer, K., Davies, D.G. & Costerton, J.W. (2002) Bio-films as complex differentiated communities. *Annual Review of Microbiology*, 56, 187–209.

Szklarczyk, D., Gable, A.L., Lyon, D., Junge, A., Wyder, S., Huerta-Cepas, J. et al. (2019) STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. *Nucleic Acids Research*, 47(D1), D607–D613.

Szklarczyk, D., Kirsch, R., Koutrouli, M., Nastou, K., Mehryary, F., Hachilif, R. et al. (2023) The STRING database in 2023: protein–protein association networks and functional enrichment analyses for any sequenced genome of interest. *Nucleic Acids Research*, 51(D1), D638–D646.

Thiel, A., Michoud, G., Moalic, Y., Flament, D. & Jebbar, M. (2014) Genetic manipulations of the hyperthermophilic piezophilic archaeon *Thermococcus barophilus*. *Applied and Environmental Microbiology*, 80(7), 2299–2306.

Van de Werken, H.J., Verhees, C.H., Akerboom, J., de Vos, W.M. & van der Oost, J. (2006) Identification of a glycolytic regulon in the archaea *Pyrococcus* and *Thermococcus*. *FEMS Microbiology Letters*, 260(1), 69–76.

Vannier, P., Marteinsson, V.T., Fridjonsson, O.H., Oger, P. & Jebbar, M. (2011) Complete genome sequence of the hyperthermophilic, piezophilic, heterotrophic, and carboxydotrophic archaeon *Thermococcus barophilus* MP. *Journal of Bacteriology*, 193(6), 1481–1482.

Vannier, P., Michoud, G., Oger, P., Marteinsson, V.P. & Jebbar, M. (2015) Genome expression of *Thermococcus barophilus* and *Thermococcus kodakarensis* in response to different hydrostatic pressure conditions. *Microbiological Research*, 166(9), 717–725.

Waskom, M.L. (2021) seaborn: statistical data visualization. *Journal of Open Source Software*, 6(60), 3021.

Whittaker, C.A. & Hynes, R.O. (2002) Distribution and evolution of von Willebrand/integrin domains: widely dispersed domains with roles in cell adhesion and elsewhere. *Molecular Biology of the Cell*, 13(10), 3369–3387.

Zeng, X., Birrien, J.L., Fouquet, Y., Cherkashov, G., Jebbar, M., Querellou, J. et al. (2009) *Pyrococcus* CH1, an obligate piezophilic hyperthermophile: extending the upper pressure-temperature limits for life. *The ISME Journal*, 3(7), 873–876.

Zhao, L., Poschmann, G., Waldera-Lupa, D., Rafiee, N., Kollmann, M. & Stühler, K. (2019) OutCyte: a novel tool for predicting unconventional protein secretion. *Scientific Reports*, 9(1), 19448.

SUPPORTING INFORMATION

Additional supporting information can be found online
in the Supporting Information section at the end of this
article.

How to cite this article: Batour, M., Laurent, S., Moalic, Y., Chamieh, H., Taha, S. & Jebbar, M. (2023) The secretome of *Thermococcus barophilus* in the presence of carbohydrates and the potential role of the TrmBL4 regulator. *Environmental Microbiology Reports*, 15(6), 530–544. Available from: https://doi.org/10.1111/1758-2229.13186