Background: Phytopathogenic fungi affecting crop and post-harvested vegetables are a major threat to food production and food storage. To face these drawbacks, producers have become increasingly dependent on agrochemicals. However, intensive use of these compounds has led to the emergence of pathogen resistance and severe negative environmental impacts. There are also a number of plant diseases for which chemical solutions are ineffective or non-existent as well as an increasing demand by consumers for pesticide-free food. Thus, biological control through the use of natural antagonistic microorganisms has emerged as a promising alternative to chemical pesticides for more rational and safe crop management. Results: The genome of the plant-associated B. amyloliquefaciens GA1 was sample sequenced. Several gene clusters involved in the synthesis of biocontrol agents were detected. Four gene clusters were shown to direct the synthesis of the cyclic lipopeptides surfactin, iturin A and fengycin as well as the iron-siderophore bacillibactin. Beside these non-ribosomaly synthetised peptides, three additional gene clusters directing the synthesis of the antibacterial polyketides macrolactin, bacillaene and difficidin were identified. Mass spectrometry analysis of culture supernatants led to the identification of these secondary metabolites, hence demonstrating that the corresponding biosynthetic gene clusters are functional in strain GA1. In addition, genes encoding enzymes involved in synthesis and export of the dipeptide antibiotic bacilysin were highlighted. However, only its chlorinated derivative, chlorotetaine, could be detected in culture supernatants. On the contrary, genes involved in ribosome-dependent synthesis of bacteriocin and other antibiotic peptides were not detected as compared to the reference strain B. amyloliquefaciens FZB42. Conclusion: The production of all of these antibiotic compounds highlights B. amyloliquefaciens GA1 as a good candidate for the development of biocontrol agents.

Background: Pure bacterial strains give better yields when producing H₂than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H₂production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research.Results: Four pureClostridiumcultures, includingC. butyricumCWBI1009,C. pasteurianumDSM525,C. beijerinckiiDSM1820 andC. felsineumDSM749, and three different co-cultures composed of (1)C. pasteurianumand C.felsineum, (2)C. butyricumandC. felsineum, (3)C. butyricumandC. pasteurianum, were grown in 20 L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H₂production (sequence 1); and the H₂-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H₂yields were obtained for starch fermentations, and the highest yield of 2.91 mol H₂/ mol hexose was reported forC. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5 L biogas/ h was observed for the co-culture (1). In general co-cultures produced H₂at higher rates than the pureClostridiumcultures, without negatively affecting the H₂yields. Interestingly, all theClostridiumstrains and co-cultures were shown to utilize lactate (present in a starch-containing medium), andC. beijerinckiiwas able to re-consume formate producing additional H₂. In the second part of the study the co-culture (3) was used to produce H₂during 13 days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence ofC. pasteurianumandC. butyricumduring this fermentation.Conclusions: The four pureClostridiumstrains and the artificial co-cultures tested in this study were shown to efficiently produce H₂using glucose and starch as carbon sources. The artificial co-cultures produced H₂at higher rates than the pure strains, while the H₂yields were only slightly affected.

Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in future transportation and energy production. However, current industrial hydrogen production processes, such as steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the low overall yield obtained is a major challenge in biological H2 production. Thus, a thorough and detailed understanding of the relationships between genome content, gene expression patterns, pathway utilisation and metabolite synthesis is required to optimise the yield of biohydrogen production pathways. In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the metabolism shifts to H2 production. The growth, H2-production, and glucose-fermentation profiles were monitored in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they were pH dependent. During unregulated-pH glucose fermentation increased H2 production was associated with concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental factors may determine whether H2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or the nitrogenase. This work, contributing to the field of dark fermentative hydrogen production, provides a multidisciplinary approach for the investigation of the processes involved in the molecular H2 metabolism of clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based on genetic engineering techniques.

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation. Beta-lactamases are enzymes produced by some bacteria and are responsible for their resistance to beta-lactam antibiotics like penicillin. Among these bacteria some of them possess a beta-lactamase that is only produced at a high level when a beta-lactam antibiotic is present outside the cell. This mechanism of regulation, named beta-lactamase induction, has been described both in Gram-negative and Gram-positive bacteria. In Staphylococcus aureus and Bacillus licheniformis, two Gram-positive bacteria, their beta-lactamase is induced by homologous proteins including a membrane-bound penicillin receptor, BlaR1, and a cytoplasmic DNA-binding protein, BlaI, acting as repressor. The first step in the induction mechanism is the acylation of the extracytoplasmic domain of BlaR receptor by the beta-lactam antibiotic concomitant with the activation of the peptidase activity of its cytoplasmic domain. The activated receptor launched a cytoplasmic signal leading to the inactivation of BlaI repressor. The nature of this cytoplasmic signal is not well understood. In our study, we identified that a dipeptide issued from the bacterial cell wall is able to inactivate the BlaI repressor. It is the first time that a cell wall fragment is shown to reenter in the cytoplasm of Gram-positive bacteria to regulate gene expression.

Escherichia coli , and presumably most other gram-negative bacteria, possesses an efficient protein machinery for recycling its peptidoglycan during cell growth. The major recycled peptidoglycan product is N -acetylglucosamine-1,6-anhydro- N -acetylmuramic acid-tetrapeptide. Its uptake from the periplasm into the cytoplasm is carried out via the AmpG protein, an intrinsic membrane protein. In gram-negative bacteria carrying an ampC β-lactamase-inducible gene on their chromosomes, the induction mechanism is directly linked to peptidoglycan recycling. After identification of the different putative hydrophobic segments by computing, the AmpG topology was experimentally determined by using β-lactamase fusion. In the proposed model, AmpG contains 10 transmembrane segments and two large cytoplasmic loops.