Mapping minor QTL for increased stearic acid content in sunflower seed oil

Increased stearic acid (C18:0) content in the seed oil of sunflower would improve the oil quality for some edible uses. The sunflower line CAS-20 (C18:0 genotype Es1Es1es2es2), developed from the high C18:0 mutant line CAS-3 (C18:0 genotype es1es1es2es2; 25% C18:0), shows increased C18:0 levels in its seed oil (8.6%). The objective of this research was to map quantitative trait loci (QTL) conferring increased C18:0 content in CAS-20 in an F₂ mapping population developed from crosses between HA-89 (wild type Es1Es1Es2Es2; low C18:0) and CAS-20, which segregates independently of the macromutation Es1 controlling high C18:0 content in CAS-3. Seed oil fatty acid composition was measured in the F₂ population by gas-liquid chromatography. A genetic linkage map of 17 linkage groups (LGs) comprising 80 RFLP and 19 SSR marker loci from this population was used to identify QTL controlling fatty acid composition. Three QTL affecting C18:0 content were identified on LG3, LG11, and LG13, with all alleles for increased C18:0 content inherited from CAS-20. In total, these QTL explained 43.6% of the C18:0 phenotypic variation. Additionally, four candidate genes (two stearate desaturase genes, SAD6 and SAD17, and a FatA and a FatB thioesterase gene) were placed on the QTL map. On the basis of positional information, QTL on LG11 was suggested to be a SAD6 locus. The results presented show that increased C18:0 content in sunflower seed oil is not a simple trait, and the markers flanking these QTL constitute a powerful tool for plant breeding programs.     

Behavior of an Aeromonas hydrophila aroA Live Vaccine in Water Microcosms

Genetically modified auxotrophic mutants of different fish pathogens have been used as live vaccines in laboratory experiments, but the behavior of the strains after release into aquatic ecosystems has not been characterized. We previously constructed and characterized an aroA mutant of Aeromonas hydrophila and studied the protection afforded by this mutant as a live vaccine in rainbow trout. In this work, we describe the survival of this strain in aquatic microcosms prepared from fish water tanks. The aroA mutant disappeared rapidly in nonfiltered, nonautoclaved fish tank water, declining below detection levels after 15 days, suggesting an inhibitory effect of the autochthonous microflora of the water. When the aroA strain was used to inoculate sterilized water, its culturability was lower than that of wild-type strain A. hydrophila AG2; after long periods of incubation, aroA cells were able to enter a viable but nonculturable state. Entry into this nonculturable state was accompanied by changes in the cell morphology from rods to spheres, but the cells appeared to remain potentially viable, as assessed by the preservation of cell membrane integrity. Supplementation of the culture medium with sodium pyruvate favored the culturability and resuscitation of the two A. hydrophila strains at low temperatures (6 and 16°C). These results contribute to a better understanding of the behavior of the aroA strain in natural environments and suggest that the inactivation of the aroA gene may be beneficial for the safety of this live vaccine for aquacultures.     

Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5_NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.     

Kinetic evidence for interaction of TMPyP4 with two different G-quadruplex conformations of human telomeric DNA

Background

Stabilization of G-quadruplex helices by small ligands has attracted growing attention because they inhibit the activity of the enzyme telomerase, which is overexpressed in > 80% cancer cells. TMPyP4, one of the most studied G-quadruplex ligands, is used as a model to show that the ligands can exhibit different binding features with different conformations of a human telomeric specific sequence.

Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs.     

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, α-DsbA1 and α-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein α-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of α-DsbA1 for both dithiol oxidants (E. coli DsbA, 12%) and disulfide isomerases (E. coli DsbC, 14%) is similar. We therefore sought to establish whether α-DsbA1 is an oxidant or an isomerase based on functional activity. The purified α-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that α-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified α-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins.