Antibiotic-free selection in E. coli: new considerations for optimal design and improved production

Background  

The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains.     

Primary cilia bend LKB1 and mTOR to their will

Primary cilia are cell surface organelles that act as sensory antennae for various input signals. In a recent issue of Nature Cell Biology, Boehlke et?al. (2010) demonstrate that bending of cilia regulates cell size through a signaling pathway involving the LKB1 and mTOR kinases.     

GmSAL1 Hydrolyzes Inositol-1,4,5-Trisphosphate and Regulates Stomatal Closure in Detached Leaves and Ion Compartmentalization in Plant Cells

Inositol polyphosphatases are important regulators since they control the catabolism of phosphoinositol derivatives, which are often signaling molecules for cellular processes. Here we report on the characterization of one of their members in soybean, GmSAL1. In contrast to the substrate specificity of its Arabidopsis homologues (AtSAL1 and AtSAL2), GmSAL1 only hydrolyzes inositol-1,4,5-trisphosphate (IP₃) but not inositol-1,3,4-trisphosphate or inositol-1,4-bisphosphate.The ectopic expression of GmSAL1 in transgenic Arabidopsis thaliana led to a reduction in IP₃ signals, which was inferred from the reduction in the cytoplasmic signals of the in vivo biomarker pleckstrin homology domain–green florescent protein fusion protein and the suppression of abscisic acid-induced stomatal closure. At the cellular level, the ectopic expression of GmSAL1 in transgenic BY-2 cells enhanced vacuolar Na⁺ compartmentalization and therefore could partially alleviate salinity stress.     

A critical motif for oligomerization and chaperone activity of bacterial alpha-heat shock proteins.

Oligomerization into multimeric complexes is a prerequisite for the chaperone function of almost all alpha-crystallin type heat shock proteins (alpha-Hsp), but the molecular details of complex assembly are poorly understood. The alpha-Hsp proteins from Bradyrhizobium japonicum are suitable bacterial models for structure-function studies of these ubiquitous stress proteins. They fall into two distinct classes, A and B, display chaperone activity in vitro and form oligomers of approximately 24 subunits. We constructed 19 derivatives containing truncations or point mutations within the N- and C-terminal regions and analyzed them by gel filtration, citrate synthase assay and coaffinity purification. Truncation of more than the initial few amino acids of the N-terminal region led to the formation of distinct dimeric to octameric structures devoid of chaperone activity. In the C-terminal extension, integrity of an isoleucine-X-isoleucine (I-X-I) motif was imperative for alpha-Hsp functionality. This I-X-I motif is one of the characteristic consensus motifs of the alpha-Hsp family, and here we provide experimental evidence of its structural and functional importance. alpha-Hsp proteins lacking the C-terminal extension were inactive, but still able to form dimers. Here, we demonstrate that the central alpha-crystallin domain alone is not sufficient for dimerization. Additional residues at the end of the N-terminal region were required for the assembly of two subunits.     

Diet influences the intake target and mitochondrial functions of Drosophila melanogaster males

In this study, we examine the dietary protein to carbohydrate ratio (P:C) on the mitochondrial functions of two Drosophila melanogaster mtDNA haplotypes. We investigated multiple physiological parameters on flies fed with either 1:12 P:C or 1:3 P:C diets. Our results provide experimental evidence that a specific haplotype has a reduction of complex I activity when the flies are fed with the 1:12 P:C diet. This study is of particular importance to understand the influence of diet on mitochondrial evolution in invasive and broadly distributed species including humans.     

Chiroptical properties of anionic and neutral nickel(II) bis(dithiolene) complexes based on methyl and dimethyl-dddt ligands

Racemic and enantiopure nickel(II) bis(dithiolene) anionic and neutral complexes based on the methyl-5,6-dihydro-1,4-dithiin-2,3-dithiolate (me-dddt) and dimethyl-5,6-dihydro-1,4-dithiin-2,3-dithiolate (dm-dddt) ligands have been experimentally and theoretically investigated with a special focus on their chiroptical properties. According to the time-dependent density-functional theory (TD-DFT) calculations, the strong near-infrared absorption bands typical for such complexes are only weakly active in circular dichroism (CD), and moreover, they have opposite signs for the axial and equatorial conformations, due to the variation of the angle between the transition electric and magnetic dipole moments, thus leading to the mutual cancellation of their contributions and the absence of these bands in the experimental CD spectra. The influence of the number of stereogenic centers and of the oxidation state of the complexes on their chiroptical properties is highlighted. The solid-state structure of the complex (TMA)[Ni(rac-me-dddt)₂] (TMA = tetramethylammonium), determined by single-crystal X-ray diffraction analysis, shows a rather unusual cis arrangement of the two dithiolene ligands, with the methyl substituents adopting an axial conformation, which is not the most stable one in the gas phase.     

Identification,molecular and biochemical characterization of a novel thermoactive and thermostable glucoamylase from Thermoanaerobacter ethanolicus

Biotechnology Letters - We identified a new glucoamylase (TeGA) from Thermoanaerobacter ethanolicus, a thermophilic anaerobic bacterium. Structural studies suggest that TeGA belongs to the family...