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In bacteria, phenotypic heterogeneity in an isogenic population compensates for the lack of genetic diversity and allows concomitant multiple survival strategies when choosing only one is too risky. This powerful tactic is exploited for competence development in streptococci where only a subset of the community triggers the pheromone signaling system ComR-ComS, resulting in a bimodal activation. However, the regulatory cascade and the underlying mechanisms of this puzzling behavior remained partially understood. Here, we show that CovRS, a well-described virulence regulatory system in pathogenic streptococci, directly controls the ComRS system to generate bimodality in the gut commensal Streptococcus salivarius and the closely related species Streptococcus thermophilus. Using single-cell analysis of fluorescent reporter strains together with regulatory mutants, we revealed that the intracellular concentration of ComR determines the proportion of competent cells in the population. We also showed that this bimodal activation requires a functional positive-feedback loop acting on ComS production, as well as its exportation and reinternalization via dedicated permeases. As the intracellular ComR concentration is critical in this process, we hypothesized that an environmental sensor could control its abundance. We systematically inactivated all two-component systems and identified CovRS as a direct repression system of comR expression. Notably, we showed that the system transduces its negative regulation through CovR binding to multiple sites in the comR promoter region. Since CovRS integrates environmental stimuli, we suggest that it is the missing piece of the puzzle that connects environmental conditions to (bimodal) competence activation in salivarius streptococci.

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci.

Nowadays, the growing human population exacerbates the need for sustainable resources. Inspiration and achievements in nutrient production or human/animal health might emanate from microorganisms and their adaptive strategies. Here, we exemplify the benefits of lactic acid bacteria (LAB) for numerous biotechnological applications and showcase their natural transformability as a fast and robust method to hereditarily influence their phenotype/traits in fundamental and applied research contexts. We described the biogenesis of the transformation machinery and we analyzed the genome of hundreds of LAB strains exploitable for human needs to predict their transformation capabilities. Finally, we provide a stepwise rational path to stimulate and optimize natural transformation with standard and synthetic biology techniques. A comprehensive understanding of the molecular mechanisms driving natural transformation will facilitate and accelerate the improvement of bacteria with properties that serve broad societal interests.

Described herein is a peptide or peptidomimetic with a length of at least 6 residues comprising, consisting essentially of, or consisting of the sequence motif Xaa1-Trp-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO:1), wherein: Xaa1 represents an aromatic residue (Phe, Tyr, Trp, His), Cys or Ser; Xaa2, Xaa3 and Xaa4 represent any residue; and Xaa5 represents Gly, lie or Val.

Competence for natural transformation extensively contributes to genome evolution and the rapid adaptability of bacteria dwelling in challenging environments. In most streptococci, this process is tightly controlled by the ComRS signaling system, which is activated through the direct interaction between the (R)RNPP-type ComR sensor and XIP pheromone (mature ComS). The overall mechanism of activation and the basis of pheromone selectivity have been previously reported in Gram-positive salivarius streptococci; however, detailed 3D-remodeling of ComR leading up to its activation remains only partially understood. Here, we identified using a semi-rational mutagenesis approach two residues in the pheromone XIP that bolster ComR sensor activation by interacting with two aromatic residues of its XIP-binding pocket. Random and targeted mutagenesis of ComR revealed that the interplay between these four residues remodel a network of aromatic-aromatic interactions involved in relaxing the sequestration of the DNA-binding domain. Based on these data, we propose a comprehensive model for ComR activation based on two major conformational changes of the XIP-binding domain. Notably, the stimulation of this newly identified trigger point by a single XIP substitution resulted in higher competence and enhanced transformability, suggesting that pheromone-sensor co-evolution counter-selects for hyperactive systems in order to maintain a trade-off between competence and bacterial fitness. Overall, this study sheds new light on the ComRS activation mechanism and how it could be exploited for biotechnological and biomedical purposes.

Constantly surrounded by kin or alien organisms in nature, eukaryotes and prokaryotes developed various communication systems to coordinate adaptive multi-entity behavior. In complex and overcrowded environments, they require to discriminate relevant signals in a myriad of pheromones to execute appropriate responses. In the human gut commensal , the cytoplasmic Rgg/RNPP regulator ComR couples competence to bacteriocin-mediated predation. Here, we describe a paralogous sensor duo, ScuR and SarF, which circumvents ComR in order to disconnect these two physiological processes. We highlighted the recurring role of Rgg/RNPP in the production of antimicrobials and designed a robust genetic screen to unveil potent/optimized peptide pheromones. Further mutational and biochemical analyses dissected the modifiable selectivity toward their pheromone and operating sequences at the subtle molecular level. Additionally, our results highlight how we might mobilize antimicrobial molecules while silencing competence in endogenous populations of human microflora and temper gut disorders provoked by bacterial pathogens.

With the specter of resurgence of pathogens due to the propagation of antibiotic resistance genes, innovative antimicrobial strategies are needed. In this review, we summarize the beneficial aspects of bacteriocins, a set of miscellaneous peptide-based bacterium killers, compared to classical antibiotics, and emphasize their use in cocktails to curb emergence of new resistance. We highlight that their prey spectrum, their molecular malleability and their multiple modes of production might lead to specific and personalized treatments to prevent systemic disorders. Complementarily, we discuss how we might exploit prevailing bacterial commensals such as Streptococcus salivarius and deliberately mobilize their bacteriocin arsenal ‘on site’ to cure multi-resistant infections or finely reshape the endogenous microbiota for prophylaxis purposes.

The present invention relates to variants of cellobiohydrolase, preferably Cbh1, which have greater cellobiohydrolase activity. The invention also relates to a genetic construct, a host cell and to an enzyme composition comprising said variants. The invention further relates to a procedure for producing fermentable sugar and a procedure for producing a bioproduct, such as bioethanol, from cellulose material with the cellobiohydrolase variants, the host cell or the enzyme composition comprising said variants.

The invention relates to a host cell, preferably a Myceliophthora thermophila cell, which presents a lower expression and/or secretion of non-contributory cellulolytic enzymes, preferably where the non-contributory cellulolytic enzyme is endoglucanase 6 comprising SEQ ID NO: 2, thereby promoting the presence of contributory cellulolytic enzymes in the enzymatic cocktail synthesised by said host cell. The invention also relates to the use of said host cells and the enzymatic cocktails synthesised by said host cells for the production of fermentable sugars of biomass and a method for producing bioproducts, preferably bioethanol, comprising the use of said host cell or the composition according to the invention.

The present invention relates to cellulase variants which comprise a proteolysis-resistant binding region or linker for binding the cellulose-binding domain (CBD) and the catalytically active domain (CAD), and which also have greater cellulolytic activity. The invention also relates to a gene construct, a host cell and an enzyme composition comprising said variants. The invention further relates to a method for producing fermentable sugar and to a method for producing a bioproduct, such as bioethanol, from cellulose material with the cellulase variants, the host cell or the enzyme composition comprising said variants.

Described herein is a peptide or peptidomimetic with a length of at least 6 residues comprising, consisting essentially of, or consisting of the sequence motif Xaa1-Trp-Xaa2-Xaa3-Xaa4-Xaa5 (SEQ ID NO:1), wherein: Xaa1 represents an aromatic residue (Phe, Tyr, Trp, His), Cys or Ser; Xaa2, Xaa3 and Xaa4 represent any residue; and Xaa5 represents Gly, Ile or Val.

The present invention relates to a Myceliophthora thermophila cell, which expresses a nucleotide sequence that codifies a recombinant beta-xylosidase enzyme comprising an amino-acid sequence having at least 70% identity with SEQ ID NO: 1, an enzymatic composition comprising said cell and/or the recombinant enzyme with beta-xylosidase activity expressed by said cell, the use of this host cell, the recombinant enzyme with beta-xylosidase activity expressed by said cell or the composition for the degradation of biomass, and a method of producing biological products, preferably bioethanol, comprising the use of said host cell, the recombinant enzyme with the beta-xylosidase activity expressed by said cell or said composition.

The invention relates to: polypeptides with polysaccharide monooxygenase activity; a host cell expressing said polypeptides, preferably a Myceliophthora thermophila cell that recombinantly expresses at least one of said polypeptides; an enzymatic composition comprising at least one of said polypeptides, preferably together with other cellulolytic enzymes; the use of said host cell, of at least one of the polypeptides with polysaccharide monooxygenase activity or of the enzymatic composition for the degradation of cellulosic biomass; and a method for producing bioproducts, preferably bioethanol, comprising the use of said host cell, of at least one of the polypeptides of the invention or of the enzymatic composition of the invention.

Thermochemical pretreatment and enzymatic hydrolysis are the areas contributing most to the operational costs of second generation ethanol in lignocellulosic biorefineries. The improvement of lignocellulosic enzyme cocktails has been significant in the recent years. Although the needs for the reduction of the energy intensity and chemical consumption in the pretreatment step are well known, the reduction of the severity of the process strongly affects the enzymatic hydrolysis yield. To explore the formulation requirements of the well known cellulolytic cocktail from Myceliophthora thermophila on mild pretreated raw materials, this cocktail was tested on steam exploded corn stover without acid impregnation. The low hemicellulose yield and significant accumulation of xylobiose compared with the standard pretreated material obtained with dilute acid impregnation evidenced a clear limitation in the conversion of xylan to xylose. In order to complement the beta-xylosidase limitation, a selection of enzymes was expressed and tested in this fungus. A controlled expression of xylosidases from Aspergillus nidulans, Aspergillus fumigatus, and Fusarium oxysporum allowed recovering hemicellulose yields reached with standard acid treated material. The results underline the need of parallel development of the pretreatment process with the optimization of the formulation of the enzymatic cocktails.

ComRS is a newly identified cell-to-cell communication system in many streptococci species that activates the competence state for natural transformation, antimicrobial peptide secretion and, in some cases, biofilm formation, three processes that affect microbial population dynamics. The ComRS system includes the pheromone ComS, and the cytoplasmic effector ComR, a member of the RRNPP superfamily which displays an N-terminal DNA-binding domain (HTH) and a C-terminal TPR-domain interacting with the peptide. The weak conservation of TPR-domains, together with the strictness vs. promiscuity of diverse ComR orthologs in presence of non-cognate peptides, suggest a co-evolution between TPR-domains and their corresponding pheromones under species-specific specialization. The ComRS-DNA ternary complex crystal structure of Streptococcus thermophilus combined with a mutational analysis allowed the discrimination of residues involved in ComS binding/activation. From these results, a novel activation mechanism that was unknown for this family of regulators was proposed. In this model, transcriptional activation is prevented in absence of the peptide by sequestration of the HTH domain, while peptide interaction induces release of this sequestered domain and ComR dimerization to allow DNA binding. Moreover, crystallization of ComRS also disclosed precious information about peptide-TPR domain recognition, highlighting regions likely to be involved in specificity. In order to verify their implication, we have tested competence activation by chimeric ComRs (i.e. hybrid proteins between ComR orthologs of S.thermophilus and S.vestibularis). Strict ComR of S. vestibularis was chosen due to the role of this species as opportunistic human pathogen. Currently, several mutants with altered peptide recognition are under investigation. This study will provide the structural basis necessary for the rational design of inhibitors, and will contribute to develop antimicrobial strategies for the

Not Applicable

The invention relates to a host cell, preferably a Myceliophthora thermophila cell, which presents a lower expression and/or secretion of non-contributory cellulolytic enzymes, preferably wherein the non-contributory cellulolytic enzyme is endoglucanase 6 comprising SEQ ID NO: 2, thereby promoting the presence of contributory cellulolytic enzymes in the enzymatic cocktail synthesised by said host cell. The invention also relates to the use of said host cells and the enzymatic cocktails synthesised by said host cells for the production of fermentable sugars of biomass, and a method for producing bioproducts, preferably bioethanol, comprising the use of said host cell or the composition according to the invention.

Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H-ThnA4-ThnA3-ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction.

The invention refers to methods and compositions for stabilizing and increasing the activity of enzymatic mixtures comprising GH61 (PMO or polysaccharide monooxigenase) polypeptides used for the degradation of cellulosic material during the saccharification step of biofuel production processes. This improvement is achieved by the addition of a nickel cation to said enzymatic mixtures before and/or during the saccharification step. Thus, the invention provides compositions comprising PMOs, cellulolytic enzymes and a nickel cation, as well as methods for preparing said compositions and methods for producing fermentable sugars and bioproducts, preferably bioethanol, from cellulosic biomass in which said compositions are used.

The present invention concerns variants of cellobiohydrolase, preferably Cbh1, which have greater cellobiohydrolase activity. The invention also relates to a gene construct, a host cell and an enzyme composition comprising said variants. The invention further relates to a method for producing fermentable sugar and a method for producing a bioproduct, such as bioethanol, from cellulose material with the cellobiohydrolase variants, the host cell or the enzyme composition comprising said variants.

Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H-ThnA4-ThnA3-ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction

The genes for tetralin (thn) utilization in Sphingomonasmacrogolitabida strain TFA are regulated at the transcriptional level by ThnR, ThnY and ThnA3. ThnR, a LysR-type transcriptional activator activates transcription specifically in response to tetralin, and ThnY is an iron-sulfur flavoprotein that may activate ThnR by protein-protein interaction. ThnA3, a Rieske-type ferredoxin that transfers electrons to the tetralin dioxygenase, prevents transcription of thn genes when the inducer molecule of the pathway is a poor substrate for the dioxygenase. The mechanism by which ThnA3 transduces this signal to the regulatory system is a major question concerning thn gene regulation. Here, we have confirmed the discriminatory function of ThnA3 and the negative role of its reduced form. We have generated ThnY variants with amino acid exchanges in the [2Fe-2S], FAD and NAD(P) H binding domains and their regulatory properties have been analyzed. Two variants, ThnY-C40S and ThnY-N201G,S206P have completely lost the discriminatory function of the regulatory system because they induced thn gene expression with different molecules such us cis-decalin, cyclohexane, trans-decalin, or benzene, which are not real inducers of the pathway. These results support a model in which ThnA3 exerts its negative modulation via the regulator ThnY.

The genes for tetralin (thn) utilization in Sphingomonas macrogolitabida strain TFA are regulated at the transcriptional level by ThnR, ThnY and ThnA3. ThnR, a LysR-type transcriptional activator activates transcription specifically in response to tetralin, and ThnY is an iron-sulfur flavoprotein that may activate ThnR by protein-protein interaction. ThnA3, a Rieske-type ferredoxin that transfers electrons to the tetralin dioxygenase, prevents transcription of thn genes when the inducer molecule of the pathway is a poor substrate for the dioxygenase. The mechanism by which ThnA3 transduces this signal to the regulatory system is a major question concerning thn gene regulation. Here, we have confirmed the discriminatory function of ThnA3 and the negative role of its reduced form. We have generated ThnY variants with amino acid exchanges in the [2Fe-2S], FAD and NAD(P) H binding domains and their regulatory properties have been analyzed. Two variants, ThnY-C40S and ThnY-N201G,S206P have completely lost the discriminatory function of the regulatory system because they induced thn gene expression with different molecules such us cis-decalin, cyclohexane, trans-decalin, or benzene, which are not real inducers of the pathway. These results support a model in which ThnA3 exerts its negative modulation via the regulator ThnY.

La tetralina es un compuesto aromático producido por la actividad industrial, y Sphingomonas macrogolitabida estirpe TFA es una bacteria Gram- que posee la capacidad de utilizar este solvente orgánico como fuente de carbono y energía durante su crecimiento aeróbico. En nuestro grupo se identificaron los genes y productos proteicos implicados en esta ruta de biodegradación. Los datos obtenidos permitieron establecer la complejidad de la regulación de los genes thn, que responde a: (i) presencia de una molécula inductora; (ii) presencia de un sustrato degradable por la dioxigenasa inicial; (iii) presencia de otra fuente de carbono alternativa preferente. En la activación de la transcripción está implicado el activador transcripcional ThnR, y ThnY, una ferredoxina reductasa de función desconocida que es esencial para la expresión de los operones thn. Además, se ha identificado a ThnA3, la ferredoxina que transfiere electrones a la dioxigenasa inicial, pero que también modula el sistema regulador. El objetivo de este trabajo de tesis consiste principalmente en estudiar la función de ThnY como regulador de la expresión de los genes thn, y elucidar cómo este co-activador anómalo ejerce su función. Por otro lado se pretende establecer el mecanismo por el cual la ferredoxina ThnA3 modula negativamente la expresión de los genes thn. Los resultados de este trabajo han permitido confirmar que a ferredoxina reductasa ThnY es imprescindible para la activación de la transcripción de los genes de degradación de la tetralina y ejerce su función mediante interacción proteína-proteína con el regulador ThnR. Además ThnY es el encargado dentro del módulo activador de recibir la información acerca de si un sustrato es metabolizable o no por esta ruta de biodegradación, señal que procede directamente de la ruta metabólica, más concretamente de la ferredoxina ThnA3. Con este complejo modelo de regulación se evita la inducción gratuita del sistema integrándose información acerca de la meta

Previous genetic studies in Sphingomonas macrogolitabida strain TFA have established that expression of genes involved in tetralin biodegradation (thn genes) requires the function of the LysR type activator ThnR and also ThnY. Sequence comparison indicated that ThnY is homologous to bacterial oxygenase-coupled NAD(P)H-dependent ferredoxin reductases. However, ThnY showed substitutions in highly conserved positions of the pyridine nucleotide binding domain of these ferredoxin reductases. ThnY expression is co-regulated with all other genes required for tetralin biodegradation, and presumably thnY is part of the thnCA3A4RY operon. ThnY has been purified, and its biochemical and functional properties were characterized. ThnY was found to be a monomeric orange-brown iron-sulfur flavoprotein (estimated mass of 37,000 Da) containing one non-covalently attached flavin adenine dinucleotide and one plant type ferredoxin 2Fe-2S cluster. It can be efficiently reduced by dithionite, but reduction by pyridine nucleotides was very poor. Consistently, ThnY-dependent reduction of cytochrome c, ferricyanide, or 2,6-dichlorophenolindophenol using NAD(P)H as the electron donor was undetectable or very weak. The addition of ThnY to electrophoretic mobility shift assays containing ThnR and a probe bearing two thn divergent promoters resulted in a 3-fold increase in protein-DNA complex formation affinity, which indicates that ThnY directly promotes thn transcription activation by ThnR.

Previous genetic studies in Sphingomonas macrogolitabida strain TFA have established that expression of genes involved in tetralin biodegradation (thn genes) requires the function of the LysR type activator ThnR and also ThnY. Sequence comparison indicated that ThnY is homologous to bacterial oxygenase-coupled NAD(P)H-dependent ferredoxin reductases. However, ThnY showed substitutions in highly conserved positions of the pyridine nucleotide binding domain of these ferredoxin reductases. ThnY expression is co-regulated with all other genes required for tetralin biodegradation, and presumably thnY is part of the thnCA3A4RY operon. ThnY has been purified, and its biochemical and functional properties were characterized. ThnY was found to be a monomeric orange-brown iron-sulfur flavoprotein (estimated mass of 37,000 Da) containing one non-covalently attached flavin adenine dinucleotide and one plant type ferredoxin 2Fe-2S cluster. It can be efficiently reduced by dithionite, but reduction by pyridine nucleotides was very poor. Consistently, ThnY-dependent reduction of cytochrome c, ferricyanide, or 2,6-dichlorophenolindophenol using NAD(P)H as the electron donor was undetectable or very weak. The addition of ThnY to electrophoretic mobility shift assays containing ThnR and a probe bearing two thn divergent promoters resulted in a 3-fold increase in protein-DNA complex formation affinity, which indicates that ThnY directly promotes thn transcription activation by ThnR.