Quorum Sensing Signaling Molecules Produced by Reference and Emerging Soft-Rot Bacteria (Dickeya and Pectobacterium spp.)

International audienceBACKGROUND: Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. METHODOLOGY/PRINCIPAL FINDINGS: Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. CONCLUSIONS/SIGNIFICANCE: Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the mechanisms of communication via the diffusible molecules. Our results designate autoinducer-1 lactones as the main targets for a global biocontrol of soft-rot bacteria communications, including those of emerging isolates

Synthesis and structural characteristics of all mono- and difluorinated 4,6-dideoxy-d-xylo-hexopyranoses

Protein–carbohydrate interactions are implicated in many biochemical/biological processes that are fundamental to life and to human health. Fluorinated carbohydrate analogues play an important role in the study of these interactions and find application as probes in chemical biology and as drugs/diagnostics in medicine. The availability and/or efficient synthesis of a wide variety of fluorinated carbohydrates is thus of great interest. Here, we report a detailed study on the synthesis of monosaccharides in which the hydroxy groups at their 4- and 6-positions are replaced by all possible mono- and difluorinated motifs. Minimization of protecting group use was a key aim. It was found that introducing electronegative substituents, either as protecting groups or as deoxygenation intermediates, was generally beneficial for increasing deoxyfluorination yields. A detailed structural study of this set of analogues demonstrated that dideoxygenation/fluorination at the 4,6-positions caused very little distortion both in the solid state and in aqueous solution. Unexpected trends in α/β anomeric ratios were identified. Increasing fluorine content always increased the α/β ratio, with very little difference between regio- or stereoisomers, except when 4,6-difluorinated

Rapid screening of diverse biotransformations for enzyme evolution

The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s per sample. The heat map output allows rapid selection of active enzymes within 96-well plates facilitating identification of industrially relevant biocatalysts. This DiBT-MS screening workflow has been applied to the directed evolution of a phenylalanine ammonia lyase (PAL) as a case study, enhancing its activity toward electron-rich cinnamic acid derivatives which are relevant to lignocellulosic biomass degradation. Additional benefits of the screening platform include the discovery of biocatalysts (kinases, imine reductases) with novel activities and the incorporation of ion mobility technology for the identification of product hits with increased confidence

Chemoenzymatic synthesis of 3-deoxy-3-fluoro-L-fucose and its enzymatic incorporation into glycoconjugates

The first synthesis of 3-deoxy-3-fluoro-L-fucose is presented, which employs a D- to L-sugar translation strategy, and involves an enzymatic oxidation of 3-deoxy-3-fluoro-L-fucitol. Enzymatic activation (FKP) and glycosylation using an a-1,2 and an a-1,3 fucosyltransferase to obtain two fluorinated trisaccharides demonstrates its potential as a novel versatile chemical probe in glycobiology

Origin of an enigmatic regional Mio-Pliocene unconformity on the Demerara plateau

The Demerara plateau, located offshore French Guiana and Suriname, is part of a passive transform continental margin particularly prone to develop slope instabilities, probably in relation to the presence of a free distal border along its steep continental slope. Slope failure occurred at different periods (Cretaceous to Neogene) and shows an overall retrogressive evolution through time. Upslope these failure headscarp, an enigmatic regional MioPliocene unconformity has been discovered through the interpretation of new academic and industrial datasets. The aim of this work is to describe and understand the origin of this surface. Our analysis shows that this unconformity is made of a series of valleys that cross-cut sedimentary strata. Each one of these valleys has a short lateral extent and is closed along two perpendicular directions, which suggests that it could correspond to a highly meandering system, or to some sub-circular depressions. The infill of these features is equivalent to the regional stratigraphic strata found outside the structures, but in a subdued position. This seems to imply that the structures have originated by a local loss of sediments at their base or by sliding processes. Furthermore, these depressions intersect each other through time, while migrating progressively downslope. We discuss a series of hypotheses that try to explain the onset and evolution of these depressions forming the Mio-Pliocene unconformity (Canyons? Slope failures? Contourite moats? Hydrate pockmarks?). Having established that these structures are depressions formed by collapse, and have many similarities with structures recently described in the literature as pockmarks associated with gas hydrate dissolution, we favor this hypothesis. We propose that these hydrate pockmarks form with a mass failure that was triggered by fluid-overpressure development at the base of the hydrate stability zone