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Xyloglucan utilization by Ruminiclostridium cellulolyticum was formerly shown to imply the uptake of large xylogluco-oligosaccharides, followed by cytosolic depolymerization into glucose, galactose, xylose, and cellobiose. This raises the question of how the anaerobic bacterium manages the simultaneous presence of multiple sugars. Using genetic and biochemical approaches targeting the corresponding metabolic pathways, we observed that, surprisingly, all sugars are catabolized, collectively, but glucose consumption is prioritized. Most selected enzymes display unusual features, especially the GTP-dependent hexokinase of glycolysis, which appeared reversible and crucial for xyloglucan utilization. In contrast, mutant strains lacking either galactokinase, cellobiose-phosphorylase, or xylulokinase still catabolize xyloglucan but display variably altered growth. Furthermore, the xylogluco-oligosaccharide depolymerization process appeared connected to the downstream pathways through an intricate network of competitive and noncompetitive inhibitions. Altogether, our data indicate that xyloglucan utilization by R. cellulolyticum relies on an energy-saving central carbon metabolism deviating from current bacterial models, which efficiently prevents carbon overflow. IMPORTANCE The study of the decomposition of recalcitrant plant biomass is of great interest as the limiting step of terrestrial carbon cycle and to produce plant-derived valuable chemicals and energy. While extracellular cellulose degradation and catabolism have been studied in detail, few publications describe the complete metabolism of hemicelluloses and, to date, the published models are limited to the extracellular degradation and sequential entry of simple sugars. Here, we describe how the model anaerobic bacterium Ruminiclostridium cellulolyticum deals with the synchronous intracellular release of glucose, galactose, xylose, and cellobiose upon cytosolic depolymerization of imported xyloglucan oligosaccharides. The described novel metabolic strategy involves the simultaneous activity of different metabolic pathways coupled to a network of inhibitions controlling the carbon flux and is distinct from the ubiquitously observed sequential uptake and metabolism of carbohydrates known as the diauxic shift. Our results highlight the diversity of cellular responses related to a complex environment.

International audience; Background: Environmental bacteria express a wide diversity of glycoside hydrolases (GH). Screening and characterization of GH from metagenomic sources provides an insight into biomass degradation strategies of non-cultivated prokaryotes. Methods: In the present report, we screened a compost metagenome for lignocellulolytic activities and identified six genes encoding enzymes belonging to family GH9 (GH9a-f). Three of these enzymes (GH9b, GH9d and GH9e) were successfully expressed and characterized. Results: A phylogenetic analysis of the catalytic domain of pro-and eukaryotic GH9 enzymes suggested the existence of two major subgroups. Bacterial GH9s displayed a wide variety of modular architectures and those harboring an N-terminal Ig-like domain, such as GH9b and GH9d, segregated from the remainder. We purified and characterized GH9 endoglucanases from both subgroups and examined their stabilities, substrate specificities and product profiles. GH9e exhibited an original hydrolysis pattern, liberating an elevated proportion of oligosaccharides longer than cellobiose. All of the enzymes exhibited processive behavior and a synergistic action on crystalline cellulose. Synergy was also evidenced between GH9d and a GH48 enzyme identified from the same metagenome. Conclusions: The characterized GH9 enzymes displayed different modular architectures and distinct substrate and product profiles. The presence of a cellulose binding domain was shown to be necessary for binding and digestion of insoluble cellulosic substrates, but not for processivity. General significance: The identification of six GH9 enzymes from a compost metagenome and the functional variety of three characterized members highlight the importance of this enzyme family in bacterial biomass deconstruction.

Ruminiclostridium cellulolyticum produces extracellular cellulosomes which contain interalia numerous family-9 glycoside hydrolases, including the inactive Cel9V. The latter shares the same organization and 79% sequence identity with the active cellulase Cel9E. Nevertheless, two aromatic residues and a four-residue stretch putatively critical for the activity are missing in Cel9V. Introduction of one Trytophan and the four-residue stretch restored some weak activity in Cel9V, whereas the replacement of its catalytic domain by that of Cel9E generated a fully active cellulase. Altogether our data indicate that a series of mutations in the catalytic domain of Cel9V lead to an essentially inactive cellulase.

International audience; The use of biosensors as sensitive and rapid alert systems is a promising perspective to monitor accidental or intentional environmental pollution, but their implementation in the field is limited by the lack of adapted inline water monitoring devices. We describe here the design and initial qualification of an analyzer prototype able to accommodate three types of biosensors based on entirely different methodologies (immunological, whole-cell, and bacteriophage biosensors), but whose responses rely on the emission of light. We developed a custom light detector and a reaction chamber compatible with the specificities of the three systems and resulting in statutory detection limits. The water analyzer prototype resulting from the COMBITOX project can be situated at level 4 on the Technology Readiness Level (TRL) scale and this technical advance paves the way to the use of biosensors on-site.

Recombination directionality factors (RDFs), or excisionases, are essential players of prophage excisive recombination. Despite the essentially catalytic role of the integrase in both integrative and excisive recombination, RDFs are required to direct the reaction towards excision and to prevent re-integration of the prophage genome when entering a lytic cycle. KplE1, HK620 and numerous (pro)phages that integrate at the same site in enterobacteria genomes (such as the argW tRNA gene) all share a highly conserved recombination module. This module comprises the attL and attR recombination sites and the RDF and integrase genes. The KplE1 RDF was named TorI after its initial identification as a negative regulator of the tor operon. However, it was characterized as an essential factor of excisive recombination. In this study, we designed an extensive random mutagenesis protocol of the torI gene and identified key residues involved in both functions of the TorI protein. We show that, in addition to TorI-TorR protein-protein interaction, TorI interacts in solution with the IntS integrase. Moreover, in vitro, TorR and IntS appear to compete for TorI binding. Finally, our mutagenesis results suggest that the C-terminal part of the TorI protein is dedicated to protein-protein interactions with both proteins TorR and IntS.

International audience; Water quality is a major safety consideration in environments that are impacted by human activity. The key challenge of the COMBITOX project is to develop a unique instrument that can accommodate several biodetector systems (see the accompanying COMBITOX papers) able to detect different pollutants such as bacteria, toxins, and heavy metals. The output signal chosen by our consortium is based on luminescence detection. Our group recently developed phage-based biosensors using gfp as a reporter gene to detect enteric bacteria in complex environments such as sea water, and the main challenge we faced was to adapt our biodetector to a luminescent signal that could fit the COMBITOX project requirements. Another key point was to use a substrate-independent reporter system in order to avoid substrate addition in the detection prototype. This paper describes the development of a phage-based biodetector using a luminescent and substrate-independent output to detect some enteric bacteria, such as Escherichia coli, in water samples. We have successfully engineered various prototypes using the HK620 and HK97 bacteriophages that use different packaging systems, and both proved functional for the integration of the full luxCDABE operon controlled by two different bacterial promoters. We show that the luxCDABE operon controlled by the PrplU bacterial promoter is the most efficient in terms of signal emission. The emission of luminescence is specific and allows the detection of 10(4) bacteria per milliliter in 1.5 h post-infection with neither a concentration nor enrichment step.

International audience; Temperate phages mediate gene transfer and can modify the properties of their host organisms through the acquisition of novel genes, a process called lysogeny. The KplE1 prophage is one of the 10 prophage regions in Escherichia coli K12 MG1655. KplE1 is defective for lysis but fully competent for site-specific recombination. The TorI recombination directionality factor is strictly required for prophage excision from the host genome. We have previously shown that DnaJ promotes KplE1 excision by increasing the affinity of TorI for its site-specific recombination DNA target. Here, we provide evidence of a direct association between TorI and DnaJ using in vitro cross-linking assays and limited proteolysis experiments that show that this interaction allows both proteins to be transiently protected from trypsin digestion. Interestingly, NMR titration experiments showed that binding of DnaJ involves specific regions of the TorI structure. These regions, mainly composed of -helices, are located on a surface opposite the DNA-binding site. Taken together, we propose that DnaJ, without the aid of DnaK/GrpE, is capable of increasing the efficiency of KplE1 excision by causing a conformational stabilization that allows TorI to adopt a more favorable conformation for binding to its specific DNA target.