# Enterococcus faecalis Uses a Phosphotransferase System permease and a host colonization-related ABC transporter for maltodextrin uptake

Nicolas Sauvageot, Abdelhamid Mokhtari, Philippe Joyet, Aurélie Budin-Verneuil, Victor S. Blancato, Guillermo D. Repizo, Céline Henry, Andreas Pikis, John Thompson, Christian Magni, et al.

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

## ► To cite this version:

Nicolas Sauvageot, Abdelhamid Mokhtari, Philippe Joyet, Aurélie Budin-Verneuil, Victor S. Blancato, et al. Enterococcus faecalis Uses a Phosphotransferase System permease and a host colonization-related ABC transporter for maltodextrin uptake. Journal of Bacteriology, 2017, 199 (9), 10.1128/JB.00878-16. hal-01606729

HAL Id: hal-01606729

https://hal.science/hal-01606729

Submitted on 25 May 2020

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_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Revised manuscript JB00878-16

Enterococcus faecalis uses a PTS permease and a host colonization-related

ABC transporter for maltodextrin uptake

Nicolas Sauvageot¹,†, Abdelhamid Mokhtari²,³,†, Philippe Joyet², Aurélie Budin-Verneuil¹,
Víctor S. Blancato⁴, Guillermo D. Repizo⁴, Céline Henry², Andreas Pikis⁵,⁶, John Thompson⁶,
Christian Magni⁴, Axel Hartke¹,*, Josef Deutscher²,⁷,*.

¹Normandie University, UNICAEN, U2RM Stress/Virulence, F-14000 Caen, France.

²Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas,
France.

³Department of Biology, 8 May 1945 University, Guelma, Algeria.

⁴Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias
Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, (S2002LRK) Rosario,
Argentina.

⁵Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring,
Maryland 20993, USA

⁶Microbial Biochemistry and Genetics Unit, Laboratory of Cell and Developmental Biology,
NIDCR, National Institutes of Health, Bethesda, MD 20892, USA.

⁷Centre National de la Recherche Scientifique, UMR8261 (affiliated with the Univ. Paris
Diderot, Sorbonne Paris Cité), Expression Génétique Microbienne, Institut de Biologie
Physico-Chimique, 75005 Paris, France.

*For correspondence. E-mail Josef.Deutscher@grignon.inra.fr; Tel. (+33) 1 30 81 54 47; Fax (+33) 1 30 81 54 57; and E-mail axel.hartke@unicaen.fr; Tel. (+33) 2 31 56 54 04; Fax: (+33) 2 31 56 53 11.

†The first two authors equally contributed to this work.

Keywords: Enterococci, maltodextrin, phosphotransferase system, ABC transporter, host colonization

Running title: Maltodextrin uptake by *Enterococcus faecalis*
33 ABSTRACT

34
35 Maltodextrin is a mixture of maltooligosaccharides, which are produced by the
36 degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-
37 linked glucose residues. Genes presumed to code for the *Enterococcus faecalis*
38 maltodextrin transporter were induced during enterococcal infection. We therefore
39 carried out a detailed study of maltodextrin transport in this organism. Depending on
40 their length (3 to 7 glucose residues), *E. faecalis* takes up maltodextrins either via MalT,
41 a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate
42 phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter
43 MdxEFG/MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the
44 PTS permease. A *malT* mutant therefore exhibits significantly reduced growth on
45 maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the
46 ABC transporter, because a *malT/mdxF* double mutant no longer grows on maltotriose.
47 The trisaccharide arrives as maltotriose-6''-P in the cell. MapP, which dephosphorylates
48 maltose-6'-P, also releases Pᵢ from maltotriose-6''-P. Maltotetraose and longer
49 maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because
50 inactivation of the membrane protein MdxF prevents growth on maltotetraose and
51 longer maltodextrins up to at least maltuheptaose. *E. faecalis* also utilizes panose and
52 isopanose and we show for the first time that in contrast to maltotriose its two isomers
53 are primarily transported via the ABC transporter. We confirm that maltodextrin
54 utilization via MdxEFG/MsmX affects the colonization capacity of *E. faecalis*, because
55 inactivation of *mdxF* significantly reduced enterococcal colonization and/or survival in
56 kidneys and liver of mice after intraperitoneal infection.
57
58

59 **IMPORTANCE** Infections by enterococci, major health-care associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as carbon and energy source was studied in *Enterococcus faecalis* and linked to its virulence.

60
61
62
63
64 Our results demonstrate that *E. faecalis* can efficiently use glycogen degradation products.
65
66
67 We show that depending on the length of the maltodextrins one of two different transporters is used: The maltose-PTS transporter MalT or the MdxEFG/MsmX ABC transporter. The latter takes up longer maltodextrins as well as complex molecules such as panose and isopanose.

68
69 INTRODUCTION

70
71 Maltose, maltotriose and higher maltodextrins represent an important carbon and
72 energy source for numerous bacteria, including several human pathogens, such as
73 *Streptococcus pyogenes* (1), *Streptococcus pneumoniae* (2), *Listeria monocytogenes* (3) and
74 *Neisseria meningitides* (4). Maltose and maltodextrins are produced by the degradation of
75 starch or glycogen and are therefore abundant in decaying plants and in the intestine and oral
76 cavity of humans and many animals. Maltose catabolism in *Enterococcus faecalis* requires the
77 proteins encoded by the five genes *malP*, *pgcM*, *malM*, *malT*, and *mapP* (5–7). The first three
78 genes are organized in an operon and they encode a maltose phosphorylase, a β-
79 phosphoglucomutase and an aldose-1-epimerase, respectively. The *malT* gene encodes a
80 maltose-specific permease belonging to the glucose family of phosphoenolpyruvate
81 (PEP):carbohydrate phosphotransferase system (PTS) transporters; it has the domain order
82 EIICBA<sup>Mal</sup> (6, 7). The *malT* gene is located upstream from *malP* but oriented in opposite
83 direction. MalT and the two general cytoplasmic PTS components EI and HPr form a
84 phosphorylation cascade in order to catalyze the uptake and phosphorylation of maltose. EI
85 autophosphorylates at a conserved histidine by using PEP as phosphoryl donor (8). The
86 phosphoryl group is then transferred via His-15 in HPr (9) and presumably His-645 in the
87 EIIA domain (10) to Cys-492 in the EIIB domain of MalT. In the last step, the P~Cys-EIIB<sup>Mal</sup>
88 domain donates its phosphoryl group to a maltose molecule bound to the membrane-spanning
89 EIIC<sup>Mal</sup> domain. Phosphorylation at the 6'-position of the disaccharide probably lowers its
90 affinity for the EIIC<sup>Mal</sup> domain and maltose-6'-P is released into the cytoplasm (11).

91 *E. faecalis* uses MapP, the gene of which is located just downstream from *malT*, to
92 dephosphorylate maltose-6'-P formed during PTS-catalyzed transport (7). Subsequently,
93 MalP phosphorolyzes intracellular maltose to α-D-glucose and glucose-1-P. The β-
94 phosphoglucomutase PgcM transforms glucose-1-P into glucose-6-P (7).

95 In addition to the PTS permease MalT, enterococci and streptococci contain an ATP
96 binding cassette (ABC) transporter, which takes up maltooligosaccharides (2, 12, 13) and in
97 some organisms also maltose (14, 15). In *E. faecalis*, the residual maltose uptake activity
98 observed for the *malT* mutant was proposed to be mediated by the ABC transporter (6, 7). The
99 maximum chain length of maltodextrins taken up via ABC transporters varies between 8 and
100 10 (16). The maltodextrin-specific ABC transporters are composed of the substrate binding
101 protein MdxE and the two integral membrane proteins MdxF and MdxG. Their genes are
102 organized in an operon (Fig. 1). The MdxEFG transporter is completed by an ATP binding
103 protein either called MalK or MsmX. MsmX serves as ATP hydrolase for other ABC
104 transport systems (17) and in contrast to *malK* its gene is usually not associated with the
105 *mdxEFG* operon. Interestingly, the *E. faecalis* genes presumed to encode MdxEFG and
106 MsmX were recently reported to be strongly induced during *E. faecalis* infection using a
107 mouse peritonitis model (18).

108 While most enterococcus and streptococcus species seem to primarily use the PTS
109 permease MalT and the maltose-6'-P phosphatase MapP for the uptake and first catabolic step
110 of maltose, the transport of maltotriose and maltotetraose, the two smallest
111 maltooligosaccharides, seems to vary from one species to another. For example, *S. pyogenes*
112 takes up maltose, maltotriose and higher maltooligosaccharides mainly via an efficient ABC
113 transport system. A mutant defective in MalE grew slowly on maltose and maltotriose (1, 19).
114 Nevertheless, slow uptake of these two compounds is catalyzed by MalT. In contrast to *S.
115 pyogenes*, the *S. mutans* PTS permease MalT (SMU.2046c) was reported to efficiently
116 transport maltose and maltotriose and to also contribute to maltotetraose uptake. Deletion of
117 *malT* prevented utilization of maltose, strongly diminished maltotriose consumption and
118 significantly slowed growth on maltotetraose (20). Finally, *S. pneumoniae* takes up maltose
119 primarily via the PTS permease MalT (SP0758), while maltotriose is transported with similar
120 efficiency by MalT and the ABC transporter MalXCD. Maltooligosaccharides are almost
121 exclusively taken up by the ABC transporter. The residual maltose uptake observed for the
122 *malT* mutant is not catalyzed by the ABC transporter (2).

123 The variations in maltotriose and maltotetraose uptake in the three described
124 streptococci, which nevertheless use the same set of transport enzymes, prompted us to carry
125 out a detailed study of maltooligosaccharide transport in *E. faecalis*. We found that
126 maltotriose is mainly taken up via the enterococcal PTS permease MalT and that maltotriose-
127 6'-P is also dephosphorylated by the phosphatase MapP. In contrast, maltotetraose and
128 higher maltodextrins are mainly taken up via the ABC transporter MdxEFG, which also
129 catalyzes the transport of panose and isopanose, two maltotriose isomers formed during
130 glycogen degradation. Interestingly, genes encoding enzymes involved in maltodextrin
131 metabolism were reported to be strongly expressed during mice soft tissue infection with *S.
132 pyogenes* (21). A similar observation was recently made during intraperitoneal infection of
133 mice with *E. faecalis* (18), suggesting that maltodextrin metabolism might be important for *E.
134 faecalis* virulence. We here confirm this hypothesis by demonstrating that deletion of the
135 ABC transporter, but not of the PTS permease MalT, significantly reduced mice organ
136 colonisation of *E. faecalis*.
137
138 MATERIALS AND METHODS

139

**Bacterial strains and growth conditions.** The *E. faecalis* wild-type strain used in this study is a maltotetraose-positive clone isolated from our stock of strain JH2-2, which is also called TX4000. The *malT* and *mdxF* mutants used in this study were derived from the maltotetraose-positive clone (Table 1). *E. faecalis* strains were routinely grown at 37°C without shaking in 100 ml sealed bottles filled with 20 to 50 ml of Luria-Bertani (LB) medium (Difco, New Jersey, USA) containing 0.5% w/v glucose. Growth studies were carried out with carbon-depleted M17MOPS (M17cc) medium, which was prepared as previously described (22). It was supplemented with 0.3% w/v of either glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, panose or isopanose. Erythromycin, chloramphenicol and tetracycline were added when appropriate at concentrations of 150 µg ml⁻¹, 10 µg ml⁻¹ and 5 µg ml⁻¹, respectively.

151 *E. coli* strains NM522 and Top10F' used for protein purification and cloning experiments, respectively (Table 1), were grown aerobically by gyratory shaking at 250 rpm in LB medium at 37°C and transformed by electroporation with a Gene Pulser apparatus (Bio-Rad Laboratories). Growth was followed by measuring the absorption at 600 nm in a Novaspec II spectrophotometer. When appropriate, erythromycin was added at a final concentration of 150 µg ml⁻¹.

157 The *Lactococcus lactis* strain IL1403 was used as host during plasmid construction for complementation experiments and was grown in M17 medium at 30°C.

159

**General molecular methods.** Restriction endonucleases, shrimp alkaline phosphatase and T4 DNA ligase were obtained from Promega and used according to the manufacturers' instructions. PCR experiments were performed with the thermocycler "Master Cycler"
Gradient" (Eppendorf) using GoTaq or Phusion DNA polymerase (Promega). The primers used for the PCR experiments are listed in Table 2. If needed, PCR products were purified using the Nucleospin® Extract II kit (Macherey-Nagel). *E. coli* was transformed by electroporation with the Gene Pulser Apparatus (Bio-Rad Laboratories), as described in (23) and *E. faecalis* or *L. lactis* as described in (24). Plasmids were extracted from *E. coli* by using the Nucleospin® Plasmid kit (Macherey-Nagel). RNA extraction was performed as previously described (25). The 5' end of the mRNA corresponding to the *mdxEFG* operon (Fig. 1) was mapped on a 5'RACE (Rapid Amplification of cDNA Ends) PCR product obtained by using the 3'/5' RACE kit (Thermofisher) and the primer race1_41965 (Table 2) for the reverse transcriptase reaction and primer race2_41965 for the PCR after poly A tailing. The PCR product was subsequently purified and sequenced with the primer race3_41965.

For the complementation of the *E. faecalis* *mdxF* mutant the wild-type *mdxF* gene was cloned into vector pAGEnt (26). In this plasmid, genes inserted at the multiple cloning site are expressed from the agmatine-inducible *aguB* promoter in the presence of 40 mM agmatine, because the plasmid contains also the gene for the repressor *AguR* of the *aguBDAC* operon. The *mdxF* gene and the plasmid were amplified with the primer pairs pAGEnt_66_forkit/pAGEnt_66_revkit and RPV_AGEnt_RBS/FRV_AGEnt_Term, respectively (Table 2). The assembly of the two PCR fragments was achieved with the NEBuilder HiFi DNA Assembly Cloning Kit (NewEngland Biolabs). *L. lactis* strain IL1403 was transformed with the resulting plasmid (named pAGEnt:66c) and subsequently plated onto GM17 medium with chloramphenicol at a final concentration of 5 µg ml⁻¹. The correct sequence of the inserted DNA fragment was confirmed for one clone and its plasmid was subsequently used to complement the *E. faecalis* *mdxF* mutant. For control experiments, empty pAGEnt was electroporated into the wild-type strain and the *mdxF* mutant.
For Quantitative real-time PCR (qRT-PCR) experiments, total RNA was extracted from maltotetraose-negative and -positive strains by using the Direct-zol™ RNA MiniPrep Kit (Zymo research). The strains were grown for 4 h in M17cc medium containing either 0.5% w/v of maltodextrin or 0.5% w/v maltodextrin and 0.5% w/v glucose. The isolated RNA was spectrophotometrically quantified and 2 µg of the total RNA was subsequently utilized for reverse transcription with the QuantiTect® Reverse Transcription kit (Qiagen). qRT-PCR was subsequently carried out with SYBR Green fluorescence using the Cfx96 Real Time PCR Machine (Bio Rad). The primers used for the qRT-PCR experiments are EF1344R and EF1344L for the *mdxF* gene and EF1345R and EF1345L for the *mdxE* gene (Table 2). Raw data were converted into expression data by the absolute quantification method using the standard curve and then normalized using the housekeeping 5S RNA gene as reference. The mean values and standard deviations were calculated from at least 3 independent experiments.

**Construction of *E. faecalis* malT and *mdxF* mutants.** *E. faecalis* mutants were constructed by introducing two stop codons at the beginning of each ORF in order to produce short truncated proteins. For the *malT* mutant, two PCR products of 1000 bp were amplified by using chromosomal DNA as template and the two primer pairs pMAD-41760-for/Mut-41760-rev and pMAD-41760-rev/Mut-41760-for, respectively (Table 2). The 5' ends of Mut-41760-rev and Mut-41760-for contain *EcoRI* restriction sites. After amplification, both PCR products were therefore cut with *EcoRI* and ligated. A second PCR was performed using the reaction mixture of the ligation as DNA template and primers pMAD-41760-for / pMAD-41760-rev. The amplified 2 kb fragment was cloned into pMAD cut with *BamHI* and *NcoI* (27) and after purification from *E. coli* Top10F' the resulting plasmid was used to transform a maltotetraose-positive *E. faecalis* JH2-2 strain according to a previously described protocol (7). After electroporation, cells were plated on GM17 agar medium containing 50 µg/ml
erythromycin and 2.35 µM X-Gal and incubated at 37°C. The presence of the plasmid in blue clones resistant to the antibiotic was verified by PCR using oligonucleotide Verif_41760 and a plasmid-based primer. To allow the second crossing over, integrants were first grown for 6 hours at 30°C and subsequently overnight at 42°C. After 2 or 3 cycles of temperature shifting, cells were plated on solid GM17 medium containing X-Gal but no antibiotics and incubated at 37°C. In several white clones the presence of the introduced *EcoRI* site in the PCR amplified *malt* gene was confirmed by restriction with this enzyme and the presence of the stop codons was confirmed by DNA sequencing. The same strategy was applied for the construction of the *mdxF* mutant, except that we used primer pairs pMAD-41966-for/Mut-41966-rev and pMAD-41966-rev/Mut-41966-for (Table 2). The two Mut primers contain *NheI* and *SpeI* restriction sites, two isoschizomeric enzymes. The correct sequence in the mutant was confirmed by PCR amplification using a specific primer of the modified sequence Verif-mut-41966 and subsequent DNA sequencing.

**Growth studies with wild-type and mutant strains.** The media used for the growth studies were inoculated with overnight cultures, which were started from the glycerol stocks kept at -20°C. Each overnight culture was washed once with one volume of physiological solution and the final ODs at 600 nm were adjusted to 2.0. A 96-well plate was filled with 200 µl M17MOPS medium supplemented with different sugars per well and inoculated with 2 µl of the washed overnight culture. Three drops of paraffin oil were added on top of the medium to avoid desiccation and to create anaerobic conditions. A Microplate Reader 680 (BioRad Laboratories) was used to follow growth at 37°C. The absorption at 595 nm was measured every 30 min over a total period of 12 to 16 hours. Growth studies were carried out at least three times and always provided very similar results.

236
237 **Purification of His-tagged *E. faecalis* maltose-6'-P phosphatase.** The maltose-6'-P
238 phosphatase of the *E. faecalis* strain JH2-2 is encoded by the *mapP* (*EFT41761*) gene. The
239 enzyme was overexpressed in *E. coli* strain NM522 from the His-tag expression vector
240 pQE30 and purified as previously described (7). The enzyme was used for dephosphorylation
241 assays of maltotriose-6''-P and maltotetraose-6'''-P as described below.

242
243 **Synthesis of malotriose-6''-P and maltotetraose-6'''-P.** Maltotriose and maltotetraose
244 phosphorylated at the O-6 position of the glucose moiety at their non-reducing end were
245 prepared enzymatically via the α-glucoside specific PTS present in palatinose-grown cells of
246 *Klebsiella pneumoniae* (28). This PTS corresponds to the α-glucoside-specific EIICB PTS
247 component with the protein id KFJ74572.1, which is encoded by the *aglB* gene present on
248 chromosome 1 of the *Klebsiella pneumoniae* type strain ATCC 13883 (29). Phosphorylated
249 derivatives of the α-linked oligosaccharides were isolated by ethanol and Ba²⁺ precipitation,
250 and further purified by ion-exchange and paper chromatography. Structures and product
251 purity were confirmed by thin-layer chromatography, mass spectrometry and NMR
252 spectroscopy.

253
254 **Mass spectrometric analysis of phosphorylated maltooligosaccharides after MapP**
255 treatment. Solutions containing maltotriose-6''-P or maltotetraose-6'''-P at a concentration of
256 2 M were prepared with 20 mM ammonium bicarbonate. Aliquots (5 µl) of these solutions
257 were added to either 40 µl of purified MapP (0.8 mg ml⁻¹, dialyzed against 20 mM ammonium
258 bicarbonate) or to 40 µl of 20 mM ammonium bicarbonate and incubated for 2 h at 37°C. The
259 samples were subsequently lyophilized and rehydrated with 10 µl of water. Aliquots of 1 µl
260 were mixed with 9 µl of a sugar matrix solution and 1 µl was spotted onto the MALDI steel
261 plate. The sugar matrix was freshly prepared and contained 100 µg/ml 2,5-dihydroxybenzoic
262 acid dissolved in a mixture of H₂O/acetonitrile/N,N-dimethylaniline (1:1:0.02 vol/vol). The
263 samples were analyzed by MALDI-TOF MS (Voyager DE super STR, AB SCIEX) by
264 irradiating them with a nitrogen laser (337 nm, 10 Hz) integrated in this instrument and by
265 recording mass spectra in the reflectron mode using a delay extraction time of 120 ns and a
266 m/z mass range between 200 and 1000 Da.

267
268 **Determination of the phosphatase activity of MapP with the malachite green assay.** To
269 follow the MapP-catalyzed dephosphorylation of maltose-6'-P, maltotriose-6''-P and
270 maltotetraose-6'''-P over various time periods we used the malachite green assay (30).
271 Dephosphorylation experiments were carried out in 650 µl assay mixtures containing 50 mM
272 Tris/HCl, pH 7.4, 5 mM MgCl₂ and 2 or 4 mM of the phosphorylated maltooligosaccharides.
273 The reaction was started by adding MapP and the samples were incubated for various time
274 periods at 37°C. The amount of MapP used in the assay was 40 µg, which allowed the
275 detection of phosphate after a few minutes incubation. Aliquots of 100 µl were withdrawn
276 after 0, 1.5, 3, 6, 10 and 20 min incubation and immediately mixed with 25 µl of the
277 malachite green reagent (30). The samples were kept for 20 min at ambient temperature
278 before 375 µl of water was added and the OD₆₃₀ determined. In Table 3, the specific activity
279 (µmol per min and mg protein) determined for the MapP-catalyzed dephosphorylation of the
280 various phospho-compounds is also expressed relative to the activity measured with maltose-
281 6'-P, which was set to 100%.

282
283 **Colonization assay of mouse organs.** The maltooligosaccharide-positive *E. faecalis* JH2-2
284 clone as well as the *mdxF*, *malt* and *tpx* mutants derived from it were grown in GM17 broth
285 at 37°C. The *tpx* mutant was used as a negative control. The *tpx* gene encodes a
286 thiolperoxidase involved in stress response and its inactivation has previously been shown to
287 lower the survival of *E. faecalis* in an LD50 mouse infection model (31). All animal
288 experiments were performed at the platform of biological resources (CURB;
289 http://icore.unicaen.fr/plateformes/curb/curb-272372.kjsp) at the University of Caen in
290 accordance with the French legislation on biomedical experimentation (Ethic authorization
291 B14118015 and N/01-09-12/20/09-15) and the European Communities Council Directive
292 from 22 september 2010 (2010/63/UE). Before being used, mice were housed at 21 ± 0.5°C,
293 in Perspex home cages with free access to food and water.

294 *E. faecalis* wild-type and mutant strains were harvested at mid-log phase and
295 resuspended in physiological solution to reach a density of ≈ 8x10⁸ CFU/ml. Male and female
296 6- to 8 weeks old Swiss white mice (CURB, France) were infected intraperitoneally with 1 ml
297 of the bacterial suspensions. The experiments were conducted with 5 mice per group. After 24
298 h of infection, mice were anesthetized by inhalation of 5% isoflurane in a mixture of 30% O₂
299 and 70% N₂O before euthanasia. A peritoneal wash was performed with 5 ml of cold
300 physiological solution and the resulting fluid was collected in sterile tubes and placed on ice
301 for 30 min. The abdomen was subsequently opened and kidneys, liver, and spleen were
302 removed and placed on ice for 30 min. The organs were crushed using pestle and mortar,
303 transferred into sterile tubes and resuspended in 3 volumes of cold physiological solution. The
304 suspensions were then homogenized by vortexing twice for 30 sec. The peritoneal fluid was
305 centrifuged (10 min at 4000 g and 4°C) and the bacterial pellet was subsequently resuspended
306 in 1 ml of cold physiological solution. Serial dilutions of all samples were plated onto GM17
307 agar in order to determine the CFU numbers. In order to determine whether there is a
308 significant difference between two groups of mice we used the Mann-Whitney test in the
309 GraphPad Prism software (GraphPad Software, San Diego, CA). A P-value < 0.05 was
310 considered to be significant. All experiments were carried out twice.
311
312 **RESULTS AND DISCUSSION**

313
314 **E. faecalis** transports maltotriose mainly via the PTS permease *MalT*. In order to
315 determine to which extent the ABC and the PTS transport systems of *E. faecalis* contribute to
316 the uptake of maltotriose, the smallest maltodextrin, we carried out growth studies with
317 mutants devoid either of the maltose-specific PTS permease *MalT* or the ABC transport
318 protein *MdxF*. We observed that the wild-type strain grew with similar efficiency on
319 maltotriose, maltose and glucose. In contrast, the *malT* mutant grew significantly slower on
320 maltose and maltotriose (Fig. 2). These results therefore suggested that *E. faecalis* transports
321 and phosphorylates the smallest maltodextrin mainly via the PTS permease *MalT*, which has
322 previously been shown to transport and phosphorylate maltose (6, 7). Indeed, a mutant deleted
323 for *mdxF*, which encodes one of the transmembrane components of the ABC transporter,
324 exhibited a growth rate on maltotriose similar to that of the wild-type strain (compare Fig. 2
325 and Fig. 3). We also constructed a *malT/mdxF* double mutant and tested its growth on the
326 three above-mentioned carbon sources. The double mutant grew normally on glucose, but was
327 no longer able to grow on maltotriose (Fig. 3), indicating that the ABC transporter
328 *MdxEFG/MsmX* is responsible for the residual growth on the trisaccharide observed for the
329 *malT* mutant (Fig. 2). In contrast, the slow growth on maltose observed for the *malT* mutant
330 (Fig. 2) was not altered in the *malT/mdxF* double mutant (Fig. 3). It therefore seems that an
331 unknown carbohydrate transporter, possibly an ABC transporter or a PTS permease, is
332 responsible for the residual growth on maltose observed for the *malT* mutant. An identical
333 observation was made for *S. pneumoniae*, where similar to the *malT* single mutant the
334 *malT/mdxE* double mutant was also able to slowly grow on maltose (2). But in contrast to *S.
335 pneumoniae*, which takes up maltotriose equally well by *MalT* and the ABC transporter, *E*.
336 *faecalis* transports the trisaccharide primarily by the PTS and only slowly by
337 MdxEFG/MsmX.

338
339 **E. faecalis** transports maltotetraose and higher maltodextrins mainly via the ABC
340 transporter **MdxEFG/MsmX**. We also tested whether **E. faecalis** transports longer
341 maltooligosaccharides ranging from maltotetraose to maltoheptaose as well as the mixture of
342 oligosaccharides present in a commercially available maltodextrin via the PTS permease
343 MalT or the ABC transporter MdxEFG/MsmX. Growth studies revealed that the wild-type
344 strain JH2-2 is able to utilize maltotetraose (Fig. 2) and maltopentaose, maltohexaose and
345 maltoheptaose (Fig. 5) as well as maltodextrin (data not shown). However, growth on
346 maltotetraose and longer maltooligosaccharides was significantly slower than growth on
347 glucose, maltose or maltotriose. Compared to JH2-2, the *mdxF* mutant grew significantly
348 slower on maltotetraose (Fig. 3), maltodextrin (data not shown) and maltopentaose (Fig. 5)
349 and did not grow at all on maltohexaose and maltoheptaose (Fig. 5). In contrast, the wild-type
350 strain and the *malT* mutant grew with identical efficiency on the above carbon sources (data
351 not shown). These results establish that *E. faecalis* takes up maltooligosaccharides composed
352 of more than three glucose residues mainly or exclusively via MdxEFG/MsmX.

353 We also complemented the *mdxF* mutant with the *mdxF* wild-type allele cloned into
354 plasmid pAGEnt, in which the inserted genes are expressed from the agmatine-inducible *aguB*
355 promoter (26). Only slight maltotetraose utilization was observed for the *mdxF* mutant
356 transformed with empty pAGEnt (Fig. 6). In contrast, when the *mdxF* mutant was transformed
357 with pAGEnt carrying the *mdxF* gene, growth on maltotetraose was restored to half the level
358 observed for the wild-type strain.

359
360 *E. faecalis* MapP dephosphorylates both, maltotriose-6''-P and maltotetraose-6'''-P.

361 Because the PTS phosphorylates its oligosaccharide substrates at the 6-position of the
362 glycosyl residue at the non-reducing end, maltotriose arrives as maltotriose-6''-P in the cell.

363 Similarly, maltose enters the cytoplasm as maltose-6'-P and due to the lack of a 6-P-α-

364 glucosidase in enterococci, it is intracellularly dephosphorylated to maltose by the enzyme

365 MapP (6, 7). To investigate whether MapP dephosphorylates the phosphorylated

366 trisaccharide, we synthesized maltotriose-6'''-P as described in Materials and Methods and

367 carried out mass spectrometry and malachite green assays. Mass spectrometry revealed that

368 MapP indeed dephosphorylates maltotriose-6'''-P. The synthesized maltotriose-6'''-P sample

369 produced a major peak corresponding to maltotriose-6'''-P carrying one Na⁺ ion (607.14 m/z),

370 a minor peak with the mass of maltotriose-6'''-P coordinated with two Na⁺ ions (629.12 m/z),

371 and a very small peak corresponding to unphosphorylated maltotriose carrying one Na⁺

372 (527.16 m/z) (Fig. 4A). After incubation for 2 h at 37°C with MapP, the peaks corresponding

373 to maltotriose-6'''-P had disappeared and a strong peak with the m/z value of

374 unphosphorylated maltotriose carrying one Na⁺ became visible (Fig. 4B).

375 By using the semiquantitative malachite green assay, which detects inorganic

376 phosphate (30), we were able to confirm that MapP efficiently dephosphorylates maltotriose-

377 6'''-P. The maximum reaction rate was about 80% of that observed with maltose-6'-P (Table

378 3). It is therefore likely that after its uptake and phosphorylation by the PTS, maltotriose-6'''-P

379 is dephosphorylated to intracellular maltotriose. Indeed, an enzyme catalyzing the hydrolysis

380 of unphosphorylated maltotriose to maltose and glucose has been identified (P. Joyet, N.

381 Sauvageot, A. Hartke and J. Deutscher, unpublished results).

382 Because MalT of *S. mutans* had been suggested to catalyze slow transport of

383 maltotetraose, we hypothesised that the slow growth of the *E. faecalis* *mdxF* mutant on

384 maltotetraose (Fig. 3) and maltopentaose (Fig. 5) might be due to their slow uptake by MalT.
In fact, only when the malT gene was also deleted, the resulting double mutant had completely lost its ability to grow on maltotetraose (Fig. 3). It was therefore likely that similar to maltose-6'-P and maltotriose-6''-P, maltotetraose-6'''-P produced during slow PTS-catalyzed uptake is also dephosphorylated by MapP in order to allow its further catabolism. We therefore synthesized also maltotetraose-6'''-P and tested its dephosphorylation by MapP. Mass spectrometry revealed that similar as observed with maltotriose-6''-P, MapP also completely dephosphorylated maltotetraose-6'''-P to maltotetraosecarrying one Na⁺. After incubation for 2 h at 37°C with MapP, the two peaks corresponding to maltotetraose-6'''-P and maltotetraose-6'''-P carrying one Na⁺ ion disappeared and a strong peak with the mass of unphosphorylated maltotetraose coordinated with one Na⁺ became visible (data not shown). According to the malachite green assay, dephosphorylation of maltotetraose-6'''-P was slightly slower than dephosphorylation of maltose-6'-P, but as fast as dephosphorylation of maltotriose-6''-P (Table 3). These results support the concept that maltotetraose is not only efficiently taken up by the maltodextrin transporter MdxEFG/MsmX, but also taken up slowly by the PTS permease MalT. In addition, the fast dephosphorylation of maltotetraose-6'''-P by MapP indicates that the rate-limiting step of the observed slow maltotetraose utilization in the *mdxF* mutant must be the MalT-catalyzed transport and phosphorylation of maltotetraose. The slow utilization of maltopentaose by the *mdxF* mutant (Fig. 5) might also be mediated by MalT. Maltotetraose (20) and maltopentaose are the longest oligosaccharides suggested to be transported by the PTS.

A spontaneous mutation in the *mdxEFG* promoter also prevents the utilization of longer maltooligosaccharides. When carrying out control experiments with *E. faecalis* JH2-2-derived mutants affected in genes not related to maltose and maltodextrin metabolism, we noticed that most of them did not grow on maltotetraose and higher maltooligosaccharides
(data not shown). By carrying out a more extensive study we observed that only 12.5% of the bacteria in our stock of *E. faecalis* JH2-2 were able to grow on maltotetraose. We isolated several maltotetraose-positive clones, grew them for about 100 generations in glucose-containing M17 medium and found that the maltotetraose-positive phenotype was stable. We subsequently sequenced the entire *mdxEFG* operon and its upstream region in several maltotetraose-positive and -negative clones and found that the only difference between them was a single base modification in the intergenic region between the *mdxE* and the *EFT4_1964* gene (Fig. 1). We determined the transcription start site of the *mdxEFG* operon by RACE PCR and found that the -10 promoter region of the *mdxEFG* operon was changed from *CATAAT* in maltotetraose-utilizing strains to *CATAAG* in strains not able to grow on maltodextrin (Fig. 1). The T for G exchange probably strongly decreases the expression of the *mdxEFG* operon and thus prevents growth on maltotetraose and higher maltooligosaccharides. qRT-PCR revealed indeed that growth on maltodextrin caused 400- to 1000-fold stronger induction of the *mdxE* and *mdxF* genes in the maltotetraose-positive strain compared to the maltotetraose-negative strain (Fig. 7). In the presence of glucose, the strong induction of *mdxE* and *mdxF* by maltodextrin was not significantly altered in the maltotetraose-positive strain, indicating that the *mdxEFG* operon is not submitted to catabolite repression (Fig. 7). To determine whether the capacity of enterococci to grow on maltooligosaccharides is rather the norm or an exception, we tested 21 different *E. faecalis* strains and found that they were all able to grow on maltotetraose (data not shown). Only the TX4000 (JH2-2) clone used for genome sequencing (see NCBI WebSite: http://www.ncbi.nlm.nih.gov/genome/808?genome_assembly_id=168551) has the *CATAAG* -10 promoter sequence and should therefore also fail to grow on maltotetraose and higher maltooligosaccharides. A comparison with the genome sequences of different *E. faecalis* strains revealed that all contain an *mdxEFG* -10 promoter sequence identical to that of the
maltotetraose-positive JH2-2 strain. Finally, it is important to mention that all mutants used in this study were derived from a maltotetraose-positive JH2-2 clone.

### E. faecalis transports panose and isopanose via the ABC transporter MdxEFG/MsmX.

Maltodextrins produced from branched chain polysaccharides, such as starch and glycogen, usually contain linkage isomers of maltotriose, such as panose and isopanose. Panose is also produced in large amounts during the degradation of pullulan by the enzyme neopullulanase (α-amylase family 13) (EC 3.2.1.135) (32). Maltotriose and the maltotriose isomers panose and isopanose differ in the linkages of the three glucose residues. In maltotriose, the three glucose residues are linked with α-1,4 glycosidic bonds. However, in panose, the first and second glucose residue (from the non-reducing end) are linked with an α-1,6 glycosidic bond.

In isopanose, the α-1,6 linkage is located between the two glucose residues at the reducing end (33). The *E. faecalis* strain V583 has been shown to grow on panose (34) and several other bacteria were reported to utilize both panose and isopanose (35–37). We found that maltotetraose-positive *E. faecalis* JH2-2 can also grow on both maltotriose isomers (Fig. 8).

However, in contrast to maltotriose, its two linkage isomers are not taken up by the PTS permease MalT, but are transported by the MdxEFG/MsmX ABC transporter. While the malT mutant grew with similar efficiency as the wild-type strain on panose and isopanose, the *mdxF* mutant was not able to utilize the two trisaccharides (Fig. 8A), indicating that the ABC transporter has a broad substrate specificity, which is not limited to α-1,4 glycosidic linear forms of polysaccharides. Therefore we propose that the function of this ABC transporter is dedicated to recover the products of degradation from starch or, in a context of infection, from glycogen.

No growth was obtained with isomaltotriose, an α-1,6-linked trisaccharide (data not shown). The main difference between maltotriose, panose or isopanose and isomaltotriose is
that the latter contains two consecutive α-1,6 linkages. Molecules with this structure might
not be recognized by the *E. faecalis* ABC transporter. Complementation of the *mdxF* mutant
with the *mdxF* wild-type allele under control of the agmatine-inducible *aguB* promoter
restored growth on panose and isopanose to about half the level observed for the wild-type
strain (Fig. 8B).

*E. faecalis* strain JH2-2 was not able to grow on β-cyclodextrin (α-1,4-linked cyclic dextrin composed of seven glucose residues) and grew only very slowly on α-cyclodextrin (composed of six glucose residues). The slow transport of α-cyclodextrin seems to be catalyzed by the ABC transporter, because only the *malF* but not the *malT* mutant had lost the capacity to slowly grow on the cyclic maltohexaose (data not shown).

**The mdxF mutant exhibits reduced colonization of mouse organs.** An increased expression of maltodextrin genes was observed during soft tissue infection with *S. pyogenes* (21). Similarly, a transcriptome study with cells of the *E. faecalis* strain V19 (a plasmid-cured derivative of strain V583) isolated from the peritoneum of infected mice had revealed that both the *mdxEFG* operon and the *msmX* gene are strongly expressed during infection. In contrast, the transcription level of the *malT* gene was not increased (18). These results suggested that the maltodextrin-specific ABC transporter might play a role in *E. faecalis* virulence. We therefore tested whether deletion of the *mdxF* gene would affect survival of *E. faecalis* in a mouse organ colonization model. A mutant deleted for the *tpx* gene, which encodes a thiolperoxidase that plays a role in enterococcal virulence (31), and the *malT* mutant served as positive and negative control strains, respectively. Twenty-four hours after intraperitoneal injection the bacterial load was determined in the peritoneum, kidneys, liver and spleen as described in Materials and Methods. No significant differences were observed between the wild-type strain and the *malT* mutant in the three organs and the peritoneum (Fig.
9A). In contrast, colonization of kidneys and liver by the *tpx* mutant was less efficient than by the wild-type strain (Fig. 9B), which confirmed its previously suggested implication in enterococcal virulence using an LD₅₀ mouse model (31). Interestingly, compared to the wild-type strain the *mdxF* mutant also exhibited significantly lower CFU numbers in kidneys (almost 5-fold lower) and in liver (16-fold lower) (Fig. 9C).

The link between carbohydrate utilization and virulence has been studied in pathogens like group A streptococci (GAS) or more recently in *S. suis* using transcriptome analyses or mouse oropharynx colonization experiments (1, 38). The combined results demonstrate that maltodextrin acquisition is likely to be a key factor in the ability of GAS to successfully infect the oropharynx. Our results extend the importance of maltodextrin metabolism to deep tissue infections. Indeed, the colonization of murine organs, especially the liver, by *E. faecalis* was less efficient in a mutant deficient in maltodextrin transport. The greatest impact on colonization was observed in the liver. This is of special interest as this organ is the storage site for glycogen, which can be degraded to maltodextrins. Glycogen released from host tissues may serve as substrate for growth of *E. faecalis*. However, cultures realized on glycogen or starch as substrates showed that *E. faecalis* JH2-2 does not grow on these polysaccharides, which is in agreement with the absence of genes related to glycogen degradation or encoding amylases in the genome of enterococci. Hence, the step between glycogen release and the formation of maltodextrins metabolizable by *E. faecalis* remains unclear. In conclusion, we demonstrate that *E. faecalis* uses the same enzymes (MalT, MapP) for efficient transport and the first catabolic step of maltose and maltotriose. MapP can also dephosphorylate maltotetraose-6'''P, suggesting that the enzyme recognizes primarily the phosphorylated glucose residue at the non-reducing end of maltooligosaccharides. While the transport system responsible for the residual uptake of maltose in the *malt* mutant remains unknown, maltotriose was found to be also slowly taken up by the MdxEFG/MsmX ABC
transporter (Fig. 10). The ABC transporter functions as the major uptake system not only for maltotetraose and longer maltooligosaccharides, but also for the maltotriose linkage isomers panose and isopanose. *E. faecalis* strain JH2-2 is not able to utilize cyclic maltodextrins. The PTS-catalyzed transport of maltose and maltotriose was found to be more efficient than the MdxEFG/MsmX-catalyzed uptake of maltotetraose and longer maltooligosaccharides. However, the utilization of maltotriose by the *malt* mutant via the ABC transporter was less efficient than that of maltotetraose, but was similarly efficient as the utilization of the two trisaccharides panose and isopanose by the wild-type strain. In conclusion, *E. faecalis* transports maltotriose and maltotetraose, which are usually present in maltodextrin, by mechanisms and transporters which strongly resemble those reported for *S. mutans* (20), but which exhibit significant differences from those reported for other streptococci, such as *S. pyogenes* (19) and *S. pneumoniae* (2). To the best of our knowledge, the transport of the maltotriose linkage isomers panose and isopanose, which are usually also present in small amounts in maltodextrin, has so far not been studied in bacteria. In *E. faecalis*, they are exclusively transported by the MdxEFG/MsmX ABC transporter.

Interestingly, while deletion of the PTS permease had no effect on *E. faecalis* virulence, inactivation of the membrane-spanning MalF significantly reduced the colonisation ability of the pathogen in kidneys and liver in a mouse infection model. With the liver and to a lesser extent the kidney being the place for storage and breakdown of glycogen, it is not surprising that inactivation of the maltodextrin ABC transporter affects the fitness of the pathogen in these organs.

531
532 ACKNOWLEDGMENTS

533
534 We thank Maria Fernandez for providing plasmid pAGEnt and Benoit Haelewyn (CURB,
535 University of Caen) for expert technical assistance for the mouse infection model.

536
537
538 FUNDING INFORMATION.

539 This work was supported by grants from the MinCyt/ECOS-Sud programme (Action N°
540 A09B03) (C.M. and J.D.), the Agencia Nacional de Promoción y Tecnológica (AN-PCyT,
541 contracts 2014-1513 (C.M.) and 2014-3482 (V.B.) Argentina), the Intramural Research
542 Program of the NIDCR, National Institutes of Health, Department of Health and Human
543 Services, Bethesda, Maryland 20892 (A.P. and J.T.), the "Initiative d'Excellence" program
544 from the French government (grant DYNAMO, ANR-11-LABX-0011) (J.D.), and by a chair
545 of excellence at the University of Caen for C.M. financed by the Region of Normandy and the
546 European Regional Development Fund (ERDF). A.M. received a fellowship from the
547 Algerian government, and V.B., G.R. and C.M. are Career Investigators of CONICET
548 (Argentina).

549
550
551
552 CONFLICTS OF INTEREST

553 The authors declare no conflicts of interest.

554
555 REFERENCES

556 1. Shelburne SA, Keith DB, Davenport MT, Horstmann N, Brennan RG, Musser JM. 2008.
557 Molecular characterization of group A *Streptococcus* maltodextrin catabolism and its
558 role in pharyngitis. Mol Microbiol 69:436–452.

559 2. Bidossi A, Mulas L, Decorosi F, Colomba L, Ricci S, Pozzi G, Deutscher J, Viti C,
560 Oggioni MR. 2012. A functional genomics approach to establish the complement of
561 carbohydrate transporters in *Streptococcus pneumoniae*. PloS One 7:e33320.

562 3. Gopal S, Berg D, Hagen N, Schriefer E-M, Stoll R, Goebel W, Kreft J. 2010. Maltose
563 and maltodextrin utilization by *Listeria monocytogenes* depend on an inducible ABC
564 transporter which is repressed by glucose. PloS One 5:e10349.

565 4. Derkaoui M, Antunes A, Nait Abdallah J, Poncet S, Mazé A, Ma Pham QM, Mokhtari
566 A, Deghmane A-E, Joyet P, Taha M-K, Deutscher J. 2016. Transport and catabolism of
567 carbohydrates by *Neisseria meningitidis*. J Mol Microbiol Biotechnol 26:320–332.

568 5. Creti R, Koch S, Fabretti F, Baldassarri L, Huebner J. 2006. Enterococcal colonization
569 of the gastro-intestinal tract: role of biofilm and environmental oligosaccharides. BMC
570 Microbiol 6:60.

571 6. Le Breton Y, Pichereau V, Sauvageot N, Auffray Y, Rincé A. 2005. Maltose utilization
572 in *Enterococcus faecalis*. J Appl Microbiol 98:806–813.

573 7. Mokhtari A, Blancato VS, Repizo GD, Henry C, Pikis A, Bourand A, de Fátima Álvarez
574 M, Immel S, Mechakra-Maza A, Hartke A, Thompson J, Magni C, Deutscher J. 2013.
575 *Enterococcus faecalis* utilizes maltose by connecting two incompatible metabolic routes
576 via a novel maltose 6'-phosphate phosphatase (MapP). Mol Microbiol 88:234–253.
577 8. Alpert CA, Frank R, Stüber K, Deutscher J, Hengstenberg W. 1985. Phosphoenolpyruvate-dependent protein kinase enzyme I of *Streptococcus faecalis*: purification and properties of the enzyme and characterization of its active center. *Biochemistry* 24:959-964.

581 9. Gassner M, Stehlik D, Schrecker O, Hengstenberg W, Maurer W, Rüterjans H. 1977. The phosphoenolpyruvate-dependent phosphotransferase system of *Staphylococcus aureus*. 2. ¹H and ³¹P-nuclear-magnetic-resonance studies on the phosphocarrier protein HPr, phosphohistidines and phosphorylated HPr. *Eur J Biochem* 75:287-296.

585 10. Dörschug M, Frank R, Kalbitzer HR, Hengstenberg W, Deutscher J. 1984. Phosphoenolpyruvate-dependent phosphorylation site in enzyme III<sup>glc</sup> of the *Escherichia coli* phosphotransferase system. *Eur J Biochem* FEBS 144:113-119.

588 11. Cao Y, Jin X, Levin EJ, Huang H, Zong Y, Quick M, Weng J, Pan Y, Love J, Punta M, Rost B, Hendrickson WA, Javitch JA, Rajashankar KR, Zhou M. 2011. Crystal structure of a phosphorylation-coupled saccharide transporter. *Nature* 473:50-54.

591 12. Puyet A, Espinosa M. 1993. Structure of the maltodextrin-uptake locus of *Streptococcus pneumoniae*. Correlation to the *Escherichia coli* maltose regulon. *J Mol Biol* 230:800-811.

594 13. Zhang X, Rogers M, Bierschenk D, Bonten MJM, Willems RJL, van Schaik W. 2013. A LacI-family regulator activates maltodextrin metabolism of *Enterococcus faecium*. *PloS One* 8:e72285.

597 14. Chen J. 2013. Molecular mechanism of the *Escherichia coli* maltose transporter. *Curr Opin Struct Biol* 23:492-498.
599 15. Monedero V, Yebra MJ, Poncet S, Deutscher J. 2008. Maltose transport in *Lactobacillus casei* and its regulation by inducer exclusion. *Res Microbiol* 159:94–102.

601 16. Ferenci T. 1980. The recognition of maltodextrins by *Escherichia coli*. *Eur J Biochem* 108:631–636.

603 17. Ferreira MJ, Sá-Nogueira I de. 2010. A multitask ATPase serving different ABC-type sugar importers in *Bacillus subtilis*. *J Bacteriol* 192:5312–5318.

605 18. Muller C, Cacaci M, Sauvageot N, Sanguinetti M, Rattei T, Eder T, Giard J-C, Kalinowski J, Hain T, Hartke A. 2015. The Intraperitoneal transcriptome of the opportunistic pathogen *Enterococcus faecalis* in mice. *PloS One* 10:e0126143.

608 19. Shelburne SA, Fang H, Okorafor N, Sumby P, Sitkiewicz I, Keith D, Patel P, Austin C, Graviss EA, Musser JM, Chow D-C. 2007. MalE of group A *Streptococcus* participates in the rapid transport of maltotriose and longer maltodextrins. *J Bacteriol* 189:2610–2617.

612 20. Webb AJ, Homer KA, Hosie AHF. 2007. A phosphoenolpyruvate-dependent phosphotransferase system is the principal maltose transporter in *Streptococcus mutans*. *J Bacteriol* 189:3322–3327.

615 21. Graham MR, Virtaneva K, Porcella SF, Gardner DJ, Long RD, Welty DM, Barry WT, Johnson CA, Parkins LD, Wright FA, Musser JM. 2006. Analysis of the transcriptome of group A *Streptococcus* in mouse soft tissue infection. *Am J Pathol* 169:927–942.

618 22. Bizzini A, Zhao C, Budin-Verneuil A, Sauvageot N, Giard J-C, Auffray Y, Hartke A. 2010. Glycerol is metabolized in a complex and strain-dependent manner in *Enterococcus faecalis*. *J Bacteriol* 192:779–785.
23. Dower WJ, Miller JF, Ragsdale CW. 1988. High efficiency transformation of *E. coli* by high voltage electroporation. Nucleic Acids Res 16:6127-6145.

24. Holo H, Nes IF. 1989. High-Frequency transformation, by electroporation, of *Lactococcus lactis* subsp. *cremoris* grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55:3119-3123.

25. Sauvageot N, Beaufils S, Mazé A, Deutscher J, Hartke A. 2006. Cloning and characterization of a gene encoding a cold-shock protein in *Lactobacillus casei*. FEMS Microbiol Lett 254:55-62.

26. Linares DM, Perez M, Ladero V, Del Rio B, Redruello B, Martin MC, Fernandez M, Alvarez MA. 2014. An agmatine-inducible system for the expression of recombinant proteins in *Enterococcus faecalis*. Microb Cell Factories 1:169.

27. Arnaud M, Chastanet A, Débarbouillé M. 2004. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl Environ Microbiol 70:6887-6891.

28. Thompson J, Robrish SA, Pikis A, Brust A, Lichtenthaler FW. 2001. Phosphorylation and metabolism of sucrose and its five linkage-isomeric α-D-glucosyl-D-fructoses by *Klebsiella pneumoniae*. Carbohydr Res 331:149-161.

29. Daligault HE, Davenport KW, Minogue TD, Bishop-Lilly KA, Bruce DC, Chain PS, Coyne SR, Frey KG, Jaissle J, Koroleva GI, Ladner JT, Lo C-C, Meincke L, Munk AC, Palacios GF, Redden CL, Johnson SL. 2014. Draft genome assembly of *Klebsiella pneumoniae* type strain ATCC 13883. Genome Announc 2: doi: 10.1128/genomeA.00939-14
30. Baykov AA, Evtushenko OA, Avaeva SM. 1988. A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. *Anal Biochem* 171:266-270.

31. La Carbona S, Sauvageot N, Giard J-C, Benachour A, Posteraro B, Auffray Y, Sanguinetti M, Hartke A. 2007. Comparative study of the physiological roles of three peroxidases (NADH peroxidase, Alkyl hydroperoxide reductase and Thiol peroxidase) in oxidative stress response, survival inside macrophages and virulence of *Enterococcus faecalis*. *Mol Microbiol* 66:1148-1163.

32. Imanaka T, Kuriki T. 1989. Pattern of action of *Bacillus stearothermophilus* neopullulanase on pullulan. *J Bacteriol* 171:369-374.

33. Hansen PI, Larsen FH, Motawia SM, Blennow A, Spraul M, Dvortsak P, Engelsen SB. 2008. Structure and hydration of the amylopectin trisaccharide building blocks--Synthesis, NMR, and molecular dynamics. *Biopolymers* 89:1179-1193.

34. Gilmore MS, Rauch M, Ramsey MM, Himes PR, Varahan S, Manson JM, Lebreton F, Hancock LE. 2015. Pheromone killing of multidrug-resistant *Enterococcus faecalis* V583 by native commensal strains. *Proc Natl Acad Sci U S A* 112:7273-7278.

35. Cha HJ, Yoon HG, Kim YW, Lee HS, Kim JW, Kweon KS, Oh BH, Park KH. 1998. Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose. *Eur J Biochem* 253:251-262.

36. Tao L, Sutcliffe IC, Russell RR, Ferretti JJ. 1993. Transport of sugars, including sucrose, by the msm transport system of *Streptococcus mutans*. *J Dent Res* 72:1386-1390.
664 37. Whiting GC, Sutcliffe IC, Russell RR. 1993. Metabolism of polysaccharides by the
665 *Streptococcus mutants dexB* gene product. J Gen Microbiol 139:2019–2026.

666 38. Ferrando ML, van Baarlen P, Orrù G, Piga R, Bongers RS, Wels M, De Greeff A, Smith
667 HE, Wells JM. 2014. Carbohydrate availability regulates virulence gene expression in
668 *Streptococcus suis*. PloS One 9:e89334.

669 39. Yagi Y, Clewell DB. 1980. Recombination-deficient mutant of *Streptococcus faecalis*. J Bacteriol 143:966–970.
670

671 40. Chopin A, Chopin MC, Moillo-Batt A, Langella P. 1984. Two plasmid-determined
672 restriction and modification systems in *Streptococcus lactis*. Plasmid 11:260–263.

673

674

675
<table><caption>TABLE 1 Bacterial strains and plasmids used in this study.</caption><thead><tr><th>Bacterial stains</th><th>Relevant Characteristics</th><th>Source or reference</th></tr></thead><tbody><tr><td colspan="3"><em>Enterococcus faecalis</em></td></tr><tr><td>JH 2-2</td><td>Fus<sup>R</sup>, Rif<sup>R</sup>, plasmid free, maltotetraose-positive wild-type strain</td><td>(39)</td></tr><tr><td><em>mdxF</em> mutant</td><td>JH2-2 carrying stop codons at the beginning of the <em>mdxF</em> gene.</td><td>This study</td></tr><tr><td><em>malt</em> mutant</td><td>JH2-2 carrying stop codons at the beginning of the <em>malt</em> gene.</td><td>This study</td></tr><tr><td>JH2-2 pAGEnt</td><td>JH2-2 harboring empty pAGEnt</td><td>This study</td></tr><tr><td><em>mdxF</em> + pAGEnt</td><td><em>mdxF</em> mutant harboring empty pAGEnt</td><td>This study</td></tr><tr><td><em>mdxF</em> + pAGEnt:66c</td><td><em>mdxF</em> mutant harboring pAGEnt with the <em>mdxF</em> gene under control of the agmatine-inducible <em>aguB</em> promoter</td><td>This study</td></tr><tr><td colspan="3"><em>Lactococcus lactis</em></td></tr><tr><td>IL1403</td><td>Wild-type strain</td><td>(40)</td></tr><tr><td colspan="3"><em>Escherichia coli</em></td></tr><tr><td>Top10F'</td><td>F'<em>[lacIq Tn10 (Tet<sup>R</sup>) mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15Δ lacX74 recA 1 araD139 Δ(ara-leu) 7697galU galK rpsL (Str<sup>R</sup>) endA1 nupG</em></td><td>Invitrogen</td></tr><tr><td>NM522[pQE30-mapP]</td><td><em>supE thi-1 D(<em>lac-proAB</em>) D(<em>mcrB-hsdSM</em>) (rk- mk-) [F'<em>proAB lacIqZ DGM15</em>] [pQE30-mapP]</td><td>(7)</td></tr><tr><td colspan="3">Plasmid s</td></tr><tr><td>pMAD</td><td>Origin pE194ts, <em>Erm<sup>R</sup>, Amp<sup>R</sup>, bgaB</em></td><td>(27)</td></tr><tr><td>pAGEnt</td><td>modified pNZ8048 plasmid, <em>Cm<sup>R</sup>, contains <em>aguR</em> <em>PaguB</em> (promoter inducible with agmatine)</td><td>(26)</td></tr><tr><td>pAGEnt:66c</td><td>pAGEnt plasmid with the <em>mdxF</em> gene under control of the agmatine-inducible <em>aguB</em> promoter</td><td>This study</td></tr></tbody></table>
681

<caption>TABLE 2 Primers used in this study.</caption>

682

<table><thead><tr><th>Primers</th><th>Sequence (5' to 3')<sup>a</sup></th><th>Orientation<sup>b</sup></th></tr></thead><tbody><tr><td>pMAD_41760_for</td><td>CCGGGCCATGGAATGAAAAAATGTTTAGTTTTG</td><td>+</td></tr><tr><td>pMAD_41760_rev</td><td>GGTGGATCCTAGTTGATTAATAACTAC</td><td>-</td></tr><tr><td>mut41760_for</td><td>CGTTAGAATTCTAGTTTATGTTTGCTG</td><td>+</td></tr><tr><td>mut41760_rev</td><td>ACATGAATTCTTACGGCTCTGTCACAC</td><td>-</td></tr><tr><td>Verif_41760</td><td>CGCCGGAAGCTAGTAAATCTTC</td><td>-</td></tr><tr><td>pMAD_41966_for</td><td>GTCAGTGGATCCTGCACCGTAGAAGTTAG</td><td>-</td></tr><tr><td>pMAD_41966_rev</td><td>AAAATCGGATCCAATTTTGGCGAAGATGGC</td><td>+</td></tr><tr><td>mut41966_for</td><td>AGGAACACTAGTGACCTAACAGCAAGGATTAG</td><td>+</td></tr><tr><td>mut41966_rev</td><td>GGGTCGCTAGCGTTCCATAGCATCATTAATGCG</td><td>-</td></tr><tr><td>Verif栏41966</td><td>TGTTAGGTCATACTAGCGTTCA</td><td>-</td></tr><tr><td>pAGEnt_66_forkit</td><td>TTTTAGGAGGAACACATCATGTTCAAGAAAAAGAAAGC</td><td>+</td></tr><tr><td>pAGEnt_66_revkit</td><td>AAGCAACACGTGCTGTAATTTTACGCCTCCTTAAATGAATTC</td><td>-</td></tr><tr><td>RPV_AGEnt_RBS</td><td>CATGATGTGTTCCTCCTAAAAG</td><td>-</td></tr><tr><td>FRV_AGEnt_Term</td><td>AATTACAGCACGTGTTGCTTT</td><td>-</td></tr><tr><td>race1_41965</td><td>TCTTTTTCAAAATCGGCGAC</td><td>-</td></tr><tr><td>race2_41965</td><td>CCCATAATTTTACGTCACCAGCTG</td><td>-</td></tr><tr><td>race3_41965</td><td>TTCGTTTTTTCTGCGCCACC</td><td>-</td></tr><tr><td>EF1344R</td><td>TCATTAATGCGTGGAAACCA</td><td>-</td></tr><tr><td>EF1344L</td><td>AAACTTCGCCAACACAAGCAGT</td><td>+</td></tr><tr><td>EF1345R</td><td>ATGCCTTCCTTTCTGGTCCT</td><td>+</td></tr><tr><td>EF1345L</td><td>CCATTGCCAAAATCAATGGT</td><td>-</td></tr></tbody></table>

684

<sup>a</sup>Underlined sequences correspond to restriction sites and bold letters indicate stop codons.

<sup>b</sup>The + means oriented in and the - oriented against the direction of transcription.

687
TABLE 3 Specific activity of MapP determined with the malachite green assay in the presence of 2 or 4 mM of its substrates maltose-6'-P, maltotriose-6''-P, and maltotetraose-6'''-P. The mean values together with the standard deviations from three independent experiments are presented.

692

<table><thead><tr><th>Substrate</th><th>Specific activity (2 mM)<br>µmol min<sup>-1</sup> mg protein<sup>-1</sup></th><th>Specific activity (4 mM)<br>µmol min<sup>-1</sup> mg protein<sup>-1</sup></th></tr></thead><tbody><tr><td>Maltose-6'-P</td><td>161 ± 15 (100%)</td><td>173 ± 28 (100%)</td></tr><tr><td>Maltotriose-6''-P</td><td>123 ± 21 (76%)</td><td>141 ± 22 (81%)</td></tr><tr><td>Maltotetraose-6'''-P</td><td>133 ± 17 (82%)</td><td>139 ± 18 (80%)</td></tr></tbody></table>

693
694 FIGURE LEGENDS

695
696 FIG 1 The *E. faecalis* JH2-2 chromosomal region containing the *mdxEFG* and the upstream
697 operon, which both encode genes required for maltodextrin utilization. (A) Presented are the
698 three genes of the *mdxEFG* operon and the three upstream genes. They encode the two
699 membrane components of the ABC transporter and the periplasmic binding protein and two
700 α-glucosidases and a glucosyl transferase, respectively (P. Joyet, N. Sauvageot, A. Hartke and
701 J. Deutscher, unpublished results). The two operons are transcribed from two divergent
702 promoters (small arrows). (B) A major part of our JH2-2 stock carries a mutation in the
703 intergenic region leading to the replacement of the T in the sixth position of the -10 promoter
704 region with a G (dashed box). The -10 promoter region of the *mdxEFG* operon is underlined,
705 the transcription initiation point is marked with a small arrow and the ribosome binding site is
706 written in italics. The start codon of *mdxE* is written in bold letters. The T to G mutation
707 prevents the utilization of maltotetraose and longer maltooligosaccharides as well as of
708 panose and isopanose. The *E. faecalis* strain V583 and all other strains, for which the genome
709 has been completely sequenced, have the CATAAT -10 promoter sequence for the *mdxEFG*
710 operon. However, these strains lack the T directly following the -10 promoter in JH2-2.
711

712 FIG 2 Growth studies with the maltotetraose-positive *E. faecalis* strain JH2-2 (filled symbols) 713 and the *malT* mutant derived from it (open symbols). Strains were grown in M17cc medium 714 supplemented with 0.3% of either glucose (◆), maltose (■), maltotriose (▲) or maltotetraose 715 (●). Growth studies were carried out as described in Materials and Methods.
716

716
717 **FIG 3** Growth studies with the *E. faecalis* *mdxF* mutant (filled symbols) and the *malT/mdxF* double mutant (open symbols). The two strains were grown in M17cc medium supplemented with 0.3% of either glucose (♦), maltose (■), maltotriose (▲) or maltotetraose (●)
718
719

720
721 **FIG 4** MapP-catalyzed dephosphorylation of maltotriose-6''-P as evidenced by mass spectrometry. Dephosphorylation assays of maltotriose-6''-P with *E. faecalis* MapP and subsequent analysis by mass spectrometry were carried out as described in Materials and Methods. (A) Untreated maltotriose-6''-P: The weak peak with a mass m/z of 527.16 corresponds to unphosphorylated maltotriose containing one Na⁺ ion. The two strongest peaks at 604.14 and 629.12 correspond to maltotriose-6''-P with one [(M+H)+Na⁺] or two [(M+H)+2Na⁺] Na⁺ ions, respectively. (B) MapP-treated maltotriose-6''-P: The two strong peaks in (A) completely disappeared after treatment with MapP and a very strong single peak appeared at 527.19, which corresponds to maltotriose carrying one Na⁺ adduct.
722
723
724
725
726
727
728
729

730
731 **FIG 5** Growth studies with the maltotetraose-positive *E. faecalis* JH2-2 strain (filled symbols) and the *mdxF* mutant derived from it (open symbols). Cells were grown in M17cc medium supplemented with 0,3% (w/v) of either maltopentaose (■), maltohexaose (◆) or maltoheptaose (●).
732
733
734

735
736 **FIG 6** Growth studies with the complemented *E. faecalis* *mdxF* mutant. The maltotetraose-positive *E. faecalis* strain JH2-2 with the empty plasmid pAGEnt (▲) and the *mdxF* mutant transformed with either the empty plasmid pAGEnt (◆) or pAGEnt carrying the *mdxF* allele (pAGEnt:66c) (■) were grown in M17cc medium supplemented with 0.3% of maltotetraose.
737
738
739

740
FIG 7 Relative transcription of the *mdxE* (black bars) and *mdxF* genes (white bars) in maltotetraose-negative (Tetra⁻) and maltotetraose-positive (Tetra⁺) JH2-2 clones. The strains were grown in M17cc medium containing either 0.5% maltodextrin or 0.5% maltodextrin plus 0.5% glucose. The mean values calculated from three independent experiments are presented. Standard deviations are indicated with vertical bars.

FIG 8 Growth studies with *E. faecalis* strains on panose and isopanose. (A) Growth was followed for the maltotetraose-positive JH2-2 strain (■), the *mdxF* (▲) and the *malT* (◆) mutants in M17cc medium supplemented with 0.3% (w/v) of either panose (open symbols) or isopanose (filled symbols). (B) The maltotetraose-positive strain JH2-2 (■) and the *mdxF* mutant derived from it (◆) were transformed with empty plasmid pADGEnt. The *mdxF* mutant was also transformed with pADGEnt:66c, which contains the *mdxF* gene under control of the agmatine-inducible *aguB* promoter (▲). The three strains were grown in M17cc medium supplemented with 0.3% (w/v) of either panose (open symbols) or isopanose (filled symbols).

FIG 9 Colonization of mouse organs by various *E. faecalis* strains. Standardized inocula of 8x10⁸ CFU of the maltotetraose-positive *E. faecalis* strain JH2-2 and either (A) the *malT*, (B) the *tpx* and (C) the *mdxF* mutants derived from it were intraperitonially injected into Swiss white mice. After 24 h (*malT* and *mdxF* mutant) or 72 h (*tpx* mutant) the survival rate was determined for each strain by bacterial counts in kidneys, liver and spleen (CFU/g tissue) and in the peritoneum (CFU/ml washing fluid). The experiments were carried out with 5 mice per group. The experiment with the *mdxF* mutant was performed twice and the results obtained with the ten mice are shown (C). The numbers obtained for each individual mouse (dots) are presented as log₁₀. The results are expressed in CFU/g (for the organs) and as CFU/ml (for the
766 peritoneal wash); mean values are presented as black bars. To compare the results obtained
767 for two different groups we used the Mann-Whitney test. The calculated *P* values of <0.05 (*)
768 and <0.005 (**) (grey horizontal bars) reflect the overall difference of the CFU values
769 determined in kidney or liver, respectively, between the groups of mice infected with the
770 *mdxF* mutant and the ones infected with the wild-type strain.

771
772 **FIG 10** Schematic presentation of the *E. faecalis* transport systems catalyzing the uptake of maltose, maltotriose, maltotetraose and the maltotriose linkage isomers panose and isopanose.
773
774 While maltose and maltotriose are primarily transported by the PTS permease MalT, maltotetraose and longer maltooligosaccharides as well as panose and isopanose are taken up
775
776 by the ABC transporter MdxEFG/MsmX. When substrates or arrows are in grey, this
777 indicates that they are transported with low efficiency. This applies for the transport of
778 maltotriose by the ABC transporter MdxEFG/MsmX and the uptake of maltose by a yet
779 unknown transport protein.
<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /></figure>
<figure><img src="image_12.png" /></figure>
<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /></figure>
<figure><img src="image_15.png" /></figure>
<figure><img src="image_16.png" /></figure>