C-terminal labelling of beta-casein

This paper is the first to report specific labelling of a native protein at its C-terminal end by carboxypeptidase Y-catalyzed transpeptidation between beta-casein and tritiated Phe amide. A tryptic digest of the radiolabelled protein was resolved by reversed-phase HPLC and a single labelled peptide was isolated therefrom. Sequence determination and FAB mass spectrometry showed that the last 2 residues (Val-209, Ile-208) of beta-casein had been deleted and Ile 207 substituted by Phe, deamidation presumably occurring after transpeptidation. Identical results were obtained by transpeptidating the isolated C-terminal tryptic heptapeptide (203-209) of native beta-casein.     

Climate‐associated genetic variation in Fagus sylvatica and potential responses to climate change in the French Alps

Local adaptation patterns have been found in many plants and animals, highlighting the genetic heterogeneity of species along their range of distribution. In the next decades, global warming is predicted to induce a change in the selective pressures that drive this adaptive variation, forcing a reshuffling of the underlying adaptive allele distributions. For species with low dispersion capacity and long generation time such as trees, the rapidity of the change could impede the migration of beneficial alleles and lower their capacity to track the changing environment. Identifying the main selective pressures driving the adaptive genetic variation is thus necessary when investigating species capacity to respond to global warming. In this study, we investigate the adaptive landscape of Fagus sylvatica along a gradient of populations in the French Alps. Using a double‐digest restriction‐site‐associated DNA (ddRAD) sequencing approach, we identified 7,000 SNPs from 570 individuals across 36 different sites. A redundancy analysis (RDA)‐derived method allowed us to identify several SNPs that were strongly associated with climatic gradients; moreover, we defined the primary selective gradients along the natural populations of F. sylvatica in the Alps. Strong effects of elevation and humidity, which contrast north‐western and south‐eastern site, were found and were believed to be important drivers of genetic adaptation. Finally, simulations of future genetic landscapes that used these findings allowed identifying populations at risk for F. sylvatica in the Alps, which could be helpful for future management plans.     

Polydnaviral Ankyrin Proteins Aid Parasitic Wasp Survival by Coordinate and Selective Inhibition of Hematopoietic and Immune NF-kappa B Signaling in Insect Hosts

Polydnaviruses are mutualists of their parasitoid wasps and express genes in immune cells of their Lepidopteran hosts. Polydnaviral genomes carry multiple copies of viral ankyrins or vankyrins. Vankyrin proteins are homologous to IκB proteins, but lack sequences for regulated degradation. We tested if Ichnoviral Vankyrins differentially impede Toll-NF-κB-dependent hematopoietic and immune signaling in a heterologous in vivo Drosophila, system. We first show that hematopoiesis and the cellular encapsulation response against parasitoid wasps are tightly-linked via NF-κB signaling. The niche, which neighbors the larval hematopoietic progenitors, responds to parasite infection. Drosophila NF-κB proteins are expressed in the niche, and non cell-autonomously influence fate choice in basal and parasite-activated hematopoiesis. These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene. I²-vank-3 and P-vank-1 differentially obstruct cellular and humoral inflammation. Additionally, their maternal expression weakens ventral embryonic patterning. We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.     

Promoter Swapping Unveils the Role of the Citrobacter rodentium CTS1 Type VI Secretion System in Interbacterial Competition

The type VI secretion system (T6SS) is a versatile secretion machine dedicated to various functions in Gram-negative bacteria, including virulence toward eukaryotic cells and antibacterial activity. Activity of T6SS might be followed in vitro by the release of two proteins, Hcp and VgrG, in the culture supernatant. Citrobacter rodentium, a rodent pathogen, harbors two T6SS gene clusters, cts1 and cts2. Reporter fusion and Hcp release assays suggested that the CTS1 T6SS was not produced or not active. The cts1 locus is composed of two divergent operons. We therefore developed a new vector allowing us to swap the two divergent endogenous promoters by P_tac and P_BAD using the λ red recombination technology. Artificial induction of both promoters demonstrated that the CTS1 T6SS is functional as shown by the Hcp release assay and confers on C. rodentium a growth advantage in antibacterial competition experiments with Escherichia coli.     

Evidence of gene flow between sympatric populations of the Middle East‐Asia Minor 1 and Mediterranean putative species of Bemisia tabaci

Bemisia tabaci is a complex of putative species that exhibit a strong geographical pattern. Crossing experiments have revealed various degrees of reproductive isolation between these nascent species, ranging between fertile first‐generation hybrids (F1) and no F1 at all. However, the relevance of these results under natural conditions is generally not known. The worldwide invasion of the putative species Middle East‐Asia Minor 1 (MEAM1) has caused secondary contacts between allopatric species, which in turn provide an opportunity to detect potential hybrids in nature. A total of 346 female B. tabaci were collected in 2003 and 2005 in the North East of Morocco and assigned to MEAM1 (119), Mediterranean (Med) (225) and a new putative species (2) using mitochondrial cytochrome oxidase (mtCOI) gene sequences. MEAM1 and Med individuals were characterized at seven microsatellite loci. MEAM1 and Med were found to be sympatric in 11 of 12 samples (6 fields/year). As previously reported from Spain, MEAM1 frequency decreased over time. The genetic data are consistent with a recent introduction of MEAM1. A Bayesian clustering analysis (Structure ) distinguished two groups, which were 100% consistent with the mtCOI groups. From several lines of evidence, two individuals were identified as hybrids. Assignment profiles using NewHybrids and allele composition indicated that they were not F1 hybrids. The results are discussed in relation to the secondary endosymbiont infection status determined on a sample of individuals, and the contrasting outcomes of the reported crossing experiments between MEAM1 and Med.     

Estrogen-Related Receptor Alpha Modulates Lactate Dehydrogenase Activity in Thyroid Tumors

Metabolic modifications of tumor cells are hallmarks of cancer. They exhibit an altered metabolism that allows them to sustain higher proliferation rates in hostile environment outside the cell. In thyroid tumors, the expression of the estrogen-related receptor α (ERRα), a major factor of metabolic adaptation, is closely related to the oxidative metabolism and the proliferative status of the cells. To elucidate the role played by ERRα in the glycolytic adaptation of tumor cells, we focused on the regulation of lactate dehydrogenases A and B (LDHA, LDHB) and the LDHA/LDHB ratio. Our study included tissue samples from 10 classical and 10 oncocytic variants of follicular thyroid tumors and 10 normal thyroid tissues, as well as samples from three human thyroid tumor cell lines: FTC-133, XTC.UC1 and RO82W-1. We identified multiple cis-acting promoter elements for ERRα, in both the LDHA and LDHB genes. The interaction between ERRα and LDH promoters was confirmed by chromatin immunoprecipitation assays and in vitro analysis for LDHB. Using knock-in and knock-out cellular models, we found an inverse correlation between ERRα expression and LDH activity. This suggests that thyroid tumor cells may reprogram their metabolic pathways through the up-regulation of ERRα by a process distinct from that proposed by the recently revisited Warburg hypothesis.     

Expression of a Yersinia pseudotuberculosis Type VI Secretion System Is Responsive to Envelope Stresses through the OmpR Transcriptional Activator

The Type VI secretion system (T6SS) is a macromolecular complex widespread in Gram-negative bacteria. Although several T6SS are required for virulence towards host models, most are necessary to eliminate competitor bacteria. Other functions, such as resistance to amoeba predation, biofilm formation or adaptation to environmental conditions have also been reported. This multitude of functions is reflected by the large repertoire of regulatory mechanisms shown to control T6SS expression, production or activation. Here, we demonstrate that one T6SS gene cluster encoded within the Yersinia pseudotuberculosis genome, T6SS-4, is regulated by OmpR, the response regulator of the two-component system EnvZ-OmpR. We first identified OmpR in a transposon mutagenesis screen. OmpR does not control the expression of the four other Y. pseudotuberculosis T6SS gene clusters and of an isolated vgrG gene, and responds to osmotic stresses to bind to and activate the T6SS-4 promoter. Finally, we show that T6SS-4 promotes Y. pseudotuberculosis survival in high osmolarity conditions and resistance to deoxycholate.     

Specificity and genetic polymorphism in the Vfm quorum sensing system of plant pathogenic bacteria of the genus Dickeya

The Vfm quorum sensing (QS) system is preponderant for the virulence of different species of the bacterial genus Dickeya. The vfm gene cluster encodes 26 genes involved in the production, sensing or transduction of the QS signal. To date, the Vfm QS signal has escaped detection by analytical chemistry methods. However, we report here a strain-specific polymorphism in the biosynthesis genes vfmO and vfmP, which is predicted to be related to the production of different analogues of the QS signal. Consequently, the Vfm communication could be impossible between strains possessing different variants of the genes vfmO/P. We constructed three Vfm QS biosensor strains possessing different vfmO/P variants and compared these biosensors for their responses to samples prepared from 34 Dickeya strains possessing different vfmO/P variants. A pattern of specificity was demonstrated, providing evidence that the polymorphism in the genes vfmO/P determines the biosynthesis of different analogues of the QS signal. Unexpectedly, this vfmO/P-dependent pattern of specificity is linked to a polymorphism in the ABC transporter gene vfmG, suggesting an adaptation of the putative permease VfmG to specifically bind different analogues of the QS signal. Accordingly, we discuss the possible involvement of VfmG as co-sensor of the Vfm two-component regulatory system.     

Genetic diversity among dairy lactococcal strains investigated by polymerase chain reaction with three arbitrary primers

Randomly amplified polymorphic DNA (RAPD) has been used for the rapid typing of Lactococcus lactis strains isolated from raw milk from the Camembert region of Normandy. It is thought that the diversity and perhaps the area strain specificity due to climatic and geographical factors of such wild-type lactococcal strains could contribute to the flavour differences and specific features detected for the same product in different areas. The patterns from 58 isolates were analysed by UPGMA dendrograms. At a similarity level of 50%, four RAPD clusters were distinguished. Clusters 1 and 2 contained strains of subspecies lactis and cluster 3 contained strains related to the C2 strain which is genetically cremoris but phenotypically lactis. The type strain of cremoris subspecies was significantly differentiated from these strains with primers P2 and P3. Thus, there was a real genetic diversity in pattern, making it possible to detect potential typical RAPD fragments.