Enzymes Required for Maltodextrin Catabolism in Enterococcus faecalis Exhibit Novel Activities

Joyet, Philippe; Mokhtari, Abdelhamid; Riboulet-Bisson, Eliette; Blancato, Víctor Sebastián; Espariz, Martín; Magni, Christian; Hartke, Axel; Deutscher, Josef; Sauvageot, Nicolas

doi:10.1128/aem.00038-17

Cited by 9 publications

(13 citation statements)

References 36 publications

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“…When searching for MalR binding sites elsewhere in the E. faecalis genome, we found two putative degenerated sites located in the promoters of physiologically related genes, which code for proteins required for the uptake and metabolism of maltotetraose and longer maltodextrins (Joyet et al, ; Sauvageot et al, ). In the closely related E. faecium E1162, the maltodextrin genes are positively regulated by an additional LacI/GalR‐type transcriptional regulator, MdxR (Zhang et al, ).…”

Section: Resultsmentioning

confidence: 99%

Enterococcus faecalis MalR acts as a repressor of the maltose operons and additionally mediates their catabolite repression via direct interaction with seryl‐phosphorylated‐HPr

Grand

Blancato

Espariz

et al. 2019

Molecular Microbiology

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Enterococci are gram‐positive pathogens and lead to cause hospital‐acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α‐polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA‐independent carbon catabolite repression. The central PTS protein P‐Ser‐HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions.

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Section: Resultsmentioning

confidence: 99%

Enterococcus faecalis MalR acts as a repressor of the maltose operons and additionally mediates their catabolite repression via direct interaction with seryl‐phosphorylated‐HPr

Grand

Blancato

Espariz

et al. 2019

Molecular Microbiology

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“…1), remained unclear. The LBA1872 gene product shares amino acid sequence similarities to GH13_31 1,6-α-glucosidases (37% identity to La GH13_31A) (21) and to a recently described disproportionating 1,4-α-glucosyltransferase from Enterococcus fecalis (MmgT; 35% identity) (25). This tentative functional assignment prompted us to express LBA1872 ( La GH13_31B) as well as the two other 1,4-α-active enzymes from the MOS gene cluster from L. acidophilus NCFM to investigate the roles of these enzymes in MOS metabolism.…”

Section: Discussionmentioning

confidence: 96%

An 1,4-α-glucosyltransferase defines a new maltodextrin catabolism scheme inLactobacillus acidophilus

Andersen¹,

Moeller²,

Poulsen

et al. 2020

Preprint

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The maltooligosaccharide (MOS) utilization locus in Lactobacillus acidophilus NCFM, a model for human small-intestine lactobacilli, encodes a family 13 subfamily 31 glycoside hydrolase (GH13_31), annotated as an 1,6-α-glucosidase. Here, we reveal that this enzyme (LaGH13_31B) is an 1,4-α-glucosyltransferase that disproportionates MOS with preference for maltotriose. LaGH13_31B acts in concert with a maltogenic α-amylase that efficiently releases maltose from MOS larger than maltotriose. Collectively, these two enzymes promote efficient conversion of preferentially odd-numbered MOS to maltose that is phosphorolysed by a maltose phosphorylase, encoded by the same locus. Structural analyses revealed the presence of a flexible elongated loop, which is unique for LaGH13_31B and its close homologues. The identified loop insertion harbours a conserved aromatic residue that modulates the activity and substrate affinity of the enzyme, thereby offering a functional signature of this previously undescribed clade, which segregates from described activities such as 1,6-α-glucosidases and sucrose isomerases within GH13_31. Sequence analyses revealed that the LaGH13_31B gene is conserved in the MOS utilization loci of lactobacilli, including acidophilus cluster members that dominate the human small intestine.IMPORTANCEThe degradation of starch in the small intestine generates short linear and branched α-glucans. The latter are poorly digestible by humans, rendering them available to the gut microbiota e.g. lactobacilli adapted to the human small intestine and considered as beneficial to health. This study unveils a previously unknown scheme of maltooligosaccharide (MOS) catabolism, via the concerted action of activity together with a classical hydrolase and a phosphorylase. The intriguing involvement of a glucosyltransferase is likely to allow fine-tuning the regulation of MOS catabolism for optimal harnessing of this key metabolic resource in the human small intestine. The study extends the suite of specificities that have been identified in GH13_31 and highlights amino acid signatures underpinning the evolution of 1,4-α-glucosyl transferases that have been recruited in the MOS catabolism pathway in lactobacilli.

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“…The cells were harvested by centrifugation. The preparation of crude extracts and the purification of His‐tagged GenR and GenA on Ni‐NTA columns under non‐denaturing conditions were carried out by following the protocol described previously (Joyet et al , ). After purification the identity of the protein was verified by mass spectrometry (Sauvageot et al , ).…”

Section: Methodsmentioning

confidence: 99%

“…The assay mixture, which also contained 1 mM of CaCl 2 , was incubated for 3 h at 37°C. Aliquots of 5 or 10 μl were spotted on thin layer silica gel 60 TLC plates (Merck) and the products formed from the various phospho‐sugars were subsequently separated as previously described (Joyet et al , ).…”

Section: Methodsmentioning

confidence: 99%

“…The glucosidases seemed to be implicated in the utilization and degradation of maltose and maltodextrin (α‐linked) and cellobiose (β‐linked). The maltose and maltodextrin utilization pathways play a role in the colonization of mice organs by E. faecalis (Sauvageot et al , ; Joyet et al , ), but no information is hitherto available for the degradation pathways of β‐glucosides.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of the gen locus involved in β‐1,6‐oligosaccharide utilization by Enterococcus faecalis

Grand

Aubourg

Pikis

et al. 2019

Molecular Microbiology

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Summary The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6‐phospho‐β‐glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of β‐1,6‐ rather than β‐1,4‐linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose‐6′‐P with a Kd of 0.04 mM and with lower affinity also other phospho‐sugars. The GenR/gentiobiose‐6′‐P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel‐shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750‐OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter.

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Enzymes Required for Maltodextrin Catabolism in Enterococcus faecalis Exhibit Novel Activities

Cited by 9 publications

References 36 publications

Enterococcus faecalis MalR acts as a repressor of the maltose operons and additionally mediates their catabolite repression via direct interaction with seryl‐phosphorylated‐HPr

Enterococcus faecalis MalR acts as a repressor of the maltose operons and additionally mediates their catabolite repression via direct interaction with seryl‐phosphorylated‐HPr

An 1,4-α-glucosyltransferase defines a new maltodextrin catabolism scheme inLactobacillus acidophilus

Characterization of the gen locus involved in β‐1,6‐oligosaccharide utilization by Enterococcus faecalis

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