Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

Background  

There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.     

HKT2;2/1, a K(+) -permeable transporter identified in a salt-tolerant rice cultivar through surveys of natural genetic polymorphism

We have investigated OsHKT2;1 natural variation in a collection of 49 cultivars with different levels of salt tolerance and geographical origins. The effect of identified polymorphism on OsHKT2;1 activity was analysed through heterologous expression of variants in Xenopus oocytes. OsHKT2;1 appeared to be a highly conserved protein with only five possible amino acid substitutions that have no substantial effect on functional properties. Our study, however, also identified a new HKT isoform, No-OsHKT2;2/1 in Nona Bokra, a highly salt-tolerant cultivar. No-OsHKT2;2/1 probably originated from a deletion in chromosome 6, producing a chimeric gene. Its 5' region corresponds to that of OsHKT2;2, whose full-length sequence is not present in Nipponbare but has been identified in Pokkali, a salt-tolerant rice cultivar. Its 3' region corresponds to that of OsHKT2;1. No-OsHKT2;2/1 is essentially expressed in roots and displays a significant level of expression at high Na(+) concentrations, in contrast to OsHKT2;1. Expressed in Xenopus oocytes or in Saccharomyces cerevisiae, No-OsHKT2;2/1 exhibited a strong permeability to Na(+) and K(+) , even at high external Na(+) concentrations, like OsHKT2;2, and in contrast to OsHKT2;1. Our results suggest that No-OsHKT2;2/1 can contribute to Nona Bokra salt tolerance by enabling root K(+) uptake under saline conditions.     

Glycerol-3-phosphate acyltransferases: Rate limiting enzymes of triacylglycerol biosynthesis

Four homologous isoforms of glycerol-3-phosphate acyltransferase (GPAT), each the product of a separate gene, catalyze the synthesis of lysophosphatidic acid from glycerol-3-phosphate and long-chain acyl-CoA. This step initiates the synthesis of all the glycerolipids and evidence from gain-of-function and loss-of-function studies in mice and in cell culture strongly suggests that each isoform contributes to the synthesis of triacylglycerol. Much work remains to fully delineate the regulation of each GPAT isoform and its individual role in triacylglycerol synthesis.     

Fabrication and Testing of Microfluidic Optomechanical Oscillators

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including microresonators. However, because of the increased acoustic radiative losses during direct liquid immersion of optomechanical devices, almost all published optomechanical experiments have been performed in solid phase. This paper discusses a recently introduced hollow microfluidic optomechanical resonator. Detailed methodology is provided to fabricate these ultra-high-Q microfluidic resonators, perform optomechanical testing, and measure radiation pressure-driven breathing mode and SBS-driven whispering gallery mode parametric vibrations. By confining liquids inside the capillary resonator, high mechanical- and optical- quality factors are simultaneously maintained.     

A Unique haplotype found in apple accessions exhibiting early bud-break could serve as a marker for breeding apples with low chilling requirements

Most commercial apple cultivars have high to medium chilling requirements and consequently are not grown in regions with warm winters. Furthermore, global climate changes raise the concern that some regions where apples are currently being produced will become unsuitable in the future. Therefore, mapping and understanding the factors governing chilling requirements are important goals towards the breeding of new apple varieties. In this study, we characterized 73 apple accessions: old local accessions, modern cultivars, and selected hybrids, all grown in the Newe Ya’ar germplasm collection in Israel under moderate winter conditions. We measured the time of vegetative bud-break as an indicator of chilling requirements and genotyped the accessions for known genetic markers and for markers we developed by re-sequencing the genome of ‘Anna’, a cultivar with very low chilling requirements. Our results show that while most of the accessions that were characterized as having early bud-break are genetically different from each other, they all share a unique haplotype in a region of ～190 kb, within a previously identified QTL for bud-break time, on chromosome 9. The alleles in the early bud-break-associated haplotype were not found in any of the late accessions tested, suggesting that the causative difference leading to the variation in bud-break time lays within or near this region, and that there is a common ancestor carrying early bud-break trait of the early accessions tested. Moreover, the markers of the unique haplotype can serve as genetic markers to accelerate the breeding of apple cultivars better adapted to warm climates.     

A molecular orbital selection approach for fast calculations of specific rotation with density functional theory

In this work, we describe a simple approach to select the most important molecular orbitals (MOs) to compute the optical rotation tensor through linear response (LR) Kohn-Sham density functional theory (KS-DFT). Taking advantage of the iterative nature of the algorithms commonly used to solve the LR equations, we select the MOs with contributions to the guess perturbed density that are larger than a certain threshold and solve the LR equations with the selected MOs only. We propose two criteria for the selection, and two definitions of the selection threshold. We then test the approach with two functionals (B3LYP and CAM-B3LYP) and two basis sets (aug-cc-pVDZ and aug-cc-pVTZ) on a set of 51 organic molecules with specific rotation spanning five orders of magnitude, 10⁰–10⁴ deg (dm⁻¹ (g/mL)⁻¹). We show that this approach indeed can provide very accurate values of specific rotation with estimated speedup that ranges from 2 to 8× with the most conservative selection criterion, and up to 20 to 30× with the intermediate criterion.     

The C2B Domain Is the Primary Ca Sensor in DOC2B: A Structural and Functional Analysis

DOC2B (double-C₂ domain) protein is thought to be a high-affinity Ca^2 + sensor for spontaneous and asynchronous neurotransmitter release. To elucidate the molecular features underlying its physiological role, we determined the crystal structures of its isolated C2A and C2B domains and examined their Ca^2 +-binding properties. We further characterized the solution structure of the tandem domains (C2AB) using small-angle X-ray scattering. In parallel, we tested structure–function correlates with live cell imaging tools. We found that, despite striking structural similarity, C2B binds Ca^2 + with considerably higher affinity than C2A. The C2AB solution structure is best modeled as two domains with a highly flexible orientation and no difference in the presence or absence of Ca^2 +. In addition, kinetic studies of C2AB demonstrate that, in the presence of unilamellar vesicles, Ca^2 + binding is stabilized, as reflected by the ~ 10-fold slower rate of Ca^2 + dissociation than in the absence of vesicles. In cells, isolated C2B translocates to the plasma membrane (PM) with an EC₅₀ of 400 nM while the C2A does not translocate at submicromolar Ca^2 + concentrations, supporting the biochemical observations. Nevertheless, C2AB translocates to the PM with an ~ 2-fold lower EC₅₀ and to a greater extent than C2B. Our results, together with previous studies, reveal that the C2B is the primary Ca^2 + sensing unit in DOC2B, whereas C2A enhances the interaction of C2AB with the PM.