Yarrowia lipolytica as a model for bio-oil production

The yeast Yarrowia lipolytica has developed very efficient mechanisms for breaking down and using hydrophobic substrates. It is considered an oleaginous yeast, based on its ability to accumulate large amounts of lipids. Completion of the sequencing of the Y. lipolytica genome and the existence of suitable tools for genetic manipulation have made it possible to use the metabolic function of this species for biotechnological applications. In this review, we describe the coordinated pathways of lipid metabolism, storage and mobilization in this yeast, focusing in particular on the roles and regulation of the various enzymes and organelles involved in these processes. The physiological responses of Y. lipolytica to hydrophobic substrates include surface-mediated and direct interfacial transport processes, the production of biosurfactants, hydrophobization of the cytoplasmic membrane and the formation of protrusions. We also discuss culture conditions, including the mode of culture control and the culture medium, as these conditions can be modified to enhance the accumulation of lipids with a specific composition and to identify links between various biological processes occurring in the cells of this yeast. Examples are presented demonstrating the potential use of Y. lipolytica in fatty-acid bioconversion, substrate valorization and single-cell oil production. Finally, this review also discusses recent progress in our understanding of the metabolic fate of hydrophobic compounds within the cell: their terminal oxidation, further degradation or accumulation in the form of intracellular lipid bodies.     

N-glycan trimming by glucosidase II is essential for Arabidopsis development

Glucosidase II, one of the early N-glycan processing enzymes and a major player in the glycoprotein folding quality control, has been described as a soluble heterodimer composed of α and β subunits. Here we present the first characterization of a plant glucosidase II α subunit at the molecular level. Expression of the Arabidopsis α subunit restored N-glycan maturation capacity in Schizosaccharomyces pombe α− or αβ−deficient mutants, but with a lower efficiency in the last case. Inactivation of the α subunit in a temperature sensitive Arabidopsis mutant blocked N-glycan processing after a first trimming by glucosidase I and strongly affected seedling development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Cecilia D’Alessio and Thomas Paccalet have equal contributions to this work An erratum to this article can be found at     

Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3′-side on a 31 nt ssDNA.The replication protein A (RPA) is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes (1–3). RPA is one of the key players in various essential processes of DNA metabolism including replication, recombination, DNA repair and telomere maintenance (1,2,4–9). The functions of this protein are based on its DNA-binding activity and specific protein–protein interactions. Its ssDNA binding properties depend on DNA length and nucleotide sequence (6,10–13). RPA is a heterotrimeric protein, composed of 70-, 32- and 14-kDa subunits, commonly referred to as RPA1, RPA2 and RPA3, respectively. There are four DNA-binding domains (DBD) located in RPA1 (DBD A, DBD B, DBD C and DBD F), one located in RPA2 (DBD D) and one belongs to RPA3 (DBD E). RPA interacts with ssDNA via four DBD: DBD A, DBD B, DBD C and DBD D (14).It is now accepted (11) that RPA binds to ssDNA in a sequential pathway with a defined polarity (15–17). RPA binds ssDNA with three different binding modes. First, binding initially involves an unstable recognition site of 8–10 nt with the high-affinity DBD A and DBD B domains on the 5′-side of the occluded ssDNA; it is designated ‘compact conformation’ or 8–10 nt binding mode. Second, this step is followed by the weaker binding of DBD C, on the 3′-side, leading to an intermediate or ‘elongated contracted’ (13–22 nt) binding mode (18–19). Finally binding of DBD D on the 3′-side forms a stable ‘elongated extended’ complex characterized by a 30 nt long occluded binding site (30 nt binding mode). Although RPA3 contains an Oligonucleotide-Binding (OB)-fold motif found in the other DBDs, there is presently no biochemical evidence that this subunit directly contacts DNA. Thus positioning of the RPA3 subunit relative to the other domains is still speculative (11,20). It has been clearly demonstrated that RPA3 is crucial for RPA function (1,2): RPA3 is involved in heterotrimer formation and is responsible for the polarity of binding to DNA (11,21,22). The scope of the data indicates that either RPA3 participates only in protein–protein interactions or that putative interaction of RPA3 with ssDNA is unstable and too transient to be detected by standard biochemical experiments. This latter possibility is likely if such interaction is provided by the 3′-side of the ssDNA, since it has been suggested that this region might be transiently accessible to the RPA DBD domains (23,24).In the past few years, thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded molecules and to identify transient contacts within nucleic acids and/or between nucleic acids and proteins, in nucleoprotein assemblies (25). Thio residues such as 4-thiothymine and 6-thioguanine absorb light at wavelengths longer than 320 nm, and thus can be selectively photo-activated. Owing to the high photo-reactivity of their triplet state, they exhibit high photo-cross-linking ability towards nucleic acid bases as well as towards amino acid residues. Here we used a combination of approaches including gel retardation assays, chemical cross-linking and cross-linking with photoreactive ssDNA probes containing 4-thiothymine, introduced at a defined site in the sequence of the ssDNA, to study interactions present in human RPA (hRPA): ssDNA complexes. These studies coupled with the identification of cross-linked targets using specific antibodies revealed that in the elongated extended hRPA:ssDNA complex RPA3 closely contacts the 3′-end positioned nucleotide and yields a covalent adduct with zero-length photolabel.     

Clustering of C-Terminal Stromal Domains of Tha4 Homo-oligomers during Translocation by the Tat Protein Transport System

The chloroplast Twin arginine translocation (Tat) pathway uses three membrane proteins and the proton gradient to transport folded proteins across sealed membranes. Precursor proteins bind to the cpTatC-Hcf106 receptor complex, triggering Tha4 assembly and protein translocation. Tha4 is required only for the translocation step and is thought to be the protein-conducting component. The organization of Tha4 oligomers was examined by substituting pairs of cysteine residues into Tha4 and inducing disulfide cross-links under varying stages of protein translocation. Tha4 formed tetramers via its transmembrane domain in unstimulated membranes and octamers in membranes stimulated by precursor and the proton gradient. Tha4 formed larger oligomers of at least 16 protomers via its carboxy tail, but such C-tail clustering only occurred in stimulated membranes. Mutational studies showed that transmembrane domain directed octamers as well as C-tail clusters require Tha4's transmembrane glutamate residue and its amphipathic helix, both of which are necessary for Tha4 function. A novel double cross-linking strategy demonstrated that both transmembrane domain directed- and C-tail directed oligomerization occur in the translocase. These results support a model in which Tha4 oligomers dock with a precursor–receptor complex and undergo a conformational switch that results in activation for protein transport. This possibly involves accretion of additional Tha4 into a larger transport-active homo-oligomer.     

Origin and evolution of the Notch signalling pathway: an overview from eukaryotic genomes

Background  

Of the 20 or so signal transduction pathways that orchestrate cell-cell interactions in metazoans, seven are involved during development. One of these is the Notch signalling pathway which regulates cellular identity, proliferation, differentiation and apoptosis via the developmental processes of lateral inhibition and boundary induction. In light of this essential role played in metazoan development, we surveyed a wide range of eukaryotic genomes to determine the origin and evolution of the components and auxiliary factors that compose and modulate this pathway.     

Fecal Calprotectin Excretion in Preterm Infants during the Neonatal Period

Background

Fecal calprotectin has been proposed as a non-invasive marker of intestinal inflammation in inflammatory bowel disease in adults and children. Fecal calprotectin levels have been reported to be much higher in both healthy full-term and preterm infants than in children and adults.

Objective

To determine the time course of fecal calprotectin (f-calprotectin) excretion in preterm infants from birth until hospital discharge and to identify factors influencing f-calprotectin levels in the first weeks of life, including bacterial establishment in the gut.

Methodology

F-calprotectin was determined using an ELISA assay in 147 samples obtained prospectively from 47 preterm infants (gestational age, and birth-weight interquartiles 27–29 weeks, and 880–1320 g, respectively) at birth, and at 2-week intervals until hospital discharge.

Principal Findings

Although median f-calprotectin excretion was 138 µg/g, a wide range of inter- and intra-individual variation in f-calprotectin values (from day 3 to day 78) was observed (86% and 67%, respectively). In multivariate regression analysis, f-calprotectin correlated negatively with ante and per natal antibiotic treatment (p = 0.001), and correlated positively with the volume of enteral feeding (mL/kg/d) (p = 0.009), the need to interrupt enteral feeding (p = 0.001), and prominent gastrointestinal colonization by Clostridium sp (p = 0.019) and Staphylococcus sp (p = 0.047).

Conclusion

During the first weeks of life, the high f-calprotectin values observed in preterm infants could be linked to the gut bacterial establishment.     

Interaction between tobramycin and CSA-13 on clinical isolates of Pseudomonas aeruginosa in a model of young and mature biofilms

The bactericidal activity of a cholic acid antimicrobial derivative, CSA-13, was tested against eight strains of Pseudomonas aeruginosa (both reference and clinical strains) and compared with the response to tobramycin. In planktonic cultures, the minimal inhibitory and minimal bactericidal concentrations of CSA-13 and tobramycin were in the 1–25 mg/L range except for one mucoid clinical strain which was much less sensitive to tobramycin (minimal bactericidal concentration, 65–125 mg/L). In young (24 h) biofilms, the sensitivity to CSA-13 was reduced (half-maximal concentration CSA-13 averaged 88 mg/L) and varied among the eight strains. The sensitivity to tobramycin was also very variable among the strains and some were fully resistant to the aminoglycoside. The combination of tobramycin with CSA-13 was synergistic in five strains. Only one strain showed antagonism between the two drugs at low concentrations of CSA-13. One reference and five clinical strains were tested in mature (12 days) biofilms. The effect of CSA-13 was delayed, some strains requiring 9 days exposure to the drug to observe a bactericidal effect. All the strains were tolerant to tobramycin but the addition of CSA-13 with tobramycin was synergistic in three strains. CSA-13 permeabilized the outer membrane of the bacteria (half-maximal concentration, 4.4 mg/L). At concentrations higher than 20 mg/L, it also permeabilized the plasma membrane of human umbilical vein endothelial cells. In conclusion, CSA-13 has bactericidal activity against P. aeruginosa even in mature biofilms and cationic steroid antibiotics can thus be considered as potential candidates for the treatment of chronic pulmonary infections of patients with cystic fibrosis. Considering its interaction with the plasma membrane of eukaryotic cells, less toxic derivatives of CSA-13 should be developed.