Like a number of anaerobic and cellulolytic Gram-positive bacteria, the model microorganism Ruminiclostridium cellulolyticum produces extracellular multi-enzymatic complexes called cellulosomes, which efficiently degrade the crystalline cellulose. Action of the complexes on cellulose releases cellobiose and longer cellodextrins but to date, little is known about the transport and utilization of the produced cellodextrins in the bacterium. A better understanding of the uptake systems and fermentation of sugars derived from cellulose could have a major impact in the field of biofuels production. We characterized a putative ABC transporter devoted to cellodextrins uptake, and a cellobiose phosphorylase (CbpA) in R. cellulolyticum. The genes encoding the components of the ABC transporter (a binding protein CuaA and two integral membrane proteins) and CbpA are expressed as a polycistronic transcriptional unit induced in the presence of cellobiose. Upstream, another polycistronic transcriptional unit encodes a two-component system (sensor and regulator), and a second binding protein CuaD, and is constitutively expressed. The products might form a three-component system inducing the expression of cuaABC and cbpA since we showed that CuaR is able to recognize the region upstream of cuaA. Biochemical analysis showed that CbpA is a strict cellobiose phosphorylase inactive on longer cellodextrins; CuaA binds to all cellodextrins (G2–G5) tested, whereas CuaD is specific to cellobiose and presents a higher affinity to this sugar. This results are in agreement with their function in transport and signalization, respectively. Characterization of a cuaD mutant, and its derivatives, indicated that the ABC transporter and CbpA are essential for growth on cellobiose and cellulose. For the first time in a Gram-positive strain, we identified a three-component system and a conjugated ABC transporter/cellobiose phosphorylase system which was shown to be essential for the growth of the model cellulolytic bacterium R. cellulolyticum on cellobiose and cellulose. This efficient and energy-saving system of transport and phosphorolysis appears to be the major cellobiose utilization pathway in R. cellulolyticum, and seems well adapted to cellulolytic life-style strain. It represents a new way to enable engineered strains to utilize cellodextrins for the production of biofuels or chemicals of interest from cellulose.

Salmonella enterica, a Gram-negative zoonotic bacterium, is mainly a food-borne pathogen and the main cause of diarrhea in humans worldwide. The main reservoirs are found in poultry farms, but they are also found in wild birds. The development of antibiotic resistance in S. enterica species raises concerns about the future of efficient therapies against this pathogen and revives the interest in bacteriophages as a useful therapy against bacterial infections. Here, we aimed to decipher and functionally annotate 10 new Salmonella phage genomes isolated in Spain in the light of phage therapy. We designed a bioinformatic pipeline using available building blocks to de novo assemble genomes and perform syntaxic annotation. We then used genome-wide analyses for taxonomic annotation enabled by vContact2 and VICTOR. We were also particularly interested in improving functional annotation using remote homologies detection and comparisons with the recently published phage-specific PHROG protein database. Finally, we searched for useful functions for phage therapy, such as systems encoded by the phage to circumvent cellular defenses with a particular focus on anti-CRISPR proteins. We, thus, were able to genetically characterize nine virulent phages and one temperate phage and identify putative functions relevant to the formulation of phage cocktails for Salmonella biocontrol.

Bacterial genome diversity is influenced by prophages, which are viral genomes integrated into the bacterial chromosome. Most prophage genes are silent but those that are expressed can provide unexpected properties to their host. Using as a modelE.coliK-12 that carries 9 defective prophages in its genome, we aimed at highlighting the impact of genes encoded by prophages on host physiology. We focused our work on AppY, a transcriptional regulator encoded on the DLP12 prophage. By performing RNA-Seq experiments, we showed that AppY production modulates the expression of more than 200 genes. Among them, 11 were identified by ChIP-Seq as direct AppY targets. AppY directly and positively regulates several genes involved in the acid stress response including the master regulator genegadEbut alsonhaRandgadY, two genes important for biofilm formation. Moreover, AppY indirectly and negatively impacts bacterial motility by favoring the degradation of FlhDC, the master regulator of the flagella biosynthesis. As a consequence of these regulatory effects, AppY increases acid stress resistance and biofilm formation while also causing a strong defect in motility. Our research shed light on the importance to consider the genetic interactions occurring between prophages and bacteria to fully understand bacterial physiology. It also highlights how a prophage-encoded transcriptional regulator integrates in a complex manner into the host regulatory network and how it benefits its host, allowing it to cope with changing environmental conditions.

The filamentous cyanobacterium Nostoc sp. strain PCC 7120 can fix N₂ when combined nitrogen is not available. Furthermore, it has to cope with reactive oxygen species generated as byproducts of photosynthesis and respiration. We have previously demonstrated the synthesis of Ser/Thr kinase Pkn22 as an important survival response of Nostoc to oxidative damage. In this study we wished to investigate the possible involvement of this kinase in signalling peroxide stress and nitrogen deprivation. Quantitative RT-PCR experiments revealed that the pkn22 gene is induced in response to peroxide stress and to combined nitrogen starvation. Electrophoretic motility assays indicated that the pkn22 promoter is recognized by the global transcriptional regulators FurA and NtcA. Transcriptomic analysis comparing a pkn22-insertion mutant and the wild type strain indicated that this kinase regulates genes involved in important cellular functions such as photosynthesis, carbon metabolism and iron acquisition. Since metabolic changes may lead to oxidative stress, we investigated whether this is the case with nitrogen starvation. Our results rather invalidate this hypothesis thereby suggesting that the function of Pkn22 under nitrogen starvation is independent of its role in response to peroxide stress. Our analyses have permitted a more complete functional description of Ser/Thr kinase in Nostoc. We have decrypted the transcriptional regulation of the pkn22 gene, and analysed the whole set of genes under the control of this kinase in response to the two environmental changes often encountered by cyanobacteria in their natural habitat: oxidative stress and nitrogen deprivation.

Despite growing awareness of their importance in soil ecology, the genetic and physiological traits of bacterial predators are still relatively poorly understood. In the course of a Myxococcus xanthus predator evolution experiment, we identified a class of genotypes leading to enhanced predation against diverse species. RNA-seq analysis demonstrated that this phenotype is linked to the constitutive activation of a predation-specific program. Functional analysis of the mutations accumulated across the evolutionary time in a two-component system and Acyl-CoA-manipulating enzymes revealed the critical roles of fatty acid metabolism and antioxidant gene induction. The former likely adapts the predator to metabolites derived from the prey while the latter protects predatory cells from reactive oxygen species generated by prey cells under stress and released upon lysis during predation. These findings reveal interesting parallels between bacterial predator–prey dynamics and pathogen–host cell interactions.

Pseudothermotoga elfii strain DSM9442 and P. elfii subsp. lettingae strain DSM14385 are hyperthermophilic bacteria. P. elfii DSM9442 is a piezophile and was isolated from a depth of over 1600 m in an oil-producing well in Africa. P. elfii subsp. lettingae is piezotolerant and was isolated from a thermophilic bioreactor fed with methanol as the sole carbon and energy source. In this study, we analyzed both strains at the genomic and transcriptomic levels, paying particular attention to changes in response to pressure increases. Transcriptomic analyses revealed common traits of adaptation to increasing hydrostatic pressure in both strains, namely, variations in transport membrane or carbohydrate metabolism, as well as species-specific adaptations such as variations in amino acid metabolism and transport for the deep P. elfii DSM9442 strain. Notably, this work highlights the central role played by the amino acid aspartate as a key intermediate of the pressure adaptation mechanisms in the deep strain P. elfii DSM9442. Our comparative genomic and transcriptomic analysis revealed a gene cluster involved in lipid metabolism that is specific to the deep strain and that was differentially expressed at high hydrostatic pressures and might, thus, be a good candidate for a piezophilic gene marker in Pseudothermotogales.