Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Selective permeability of different connexin channels to the second messenger cyclic AMP

Gap junctions are intercellular conduits that are formed in vertebrates by connexin proteins and allow diffusion exchange of intracellular ions and small molecules. At least 20 different connexin genes in the human and mouse genome are cell-type specifically expressed with overlapping expression patterns. A possible explanation for this diversity could be different permeability of biologically important molecules, such as second messenger molecules. We have recently demonstrated that cyclic nucleotide-gated channels can be used to quantify gap junction-mediated diffusion of cyclic AMP. Using this method we have compared the relative permeability of gap junction channels composed of connexin 26, 32, 36, 43, 45, or 47 proteins toward the second messenger cAMP. Here we show that cAMP permeates through the investigated connexin channels with up to 30-fold different efficacy. Our results suggest that intercellular cAMP signaling in different cell types can be affected by the connexin expression pattern.     

Proteasome activity imaging and profiling characterizes bacterial effector syringolin A

Syringolin A (SylA) is a nonribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv syringae that can inhibit the eukaryotic proteasome. The proteasome is a multisubunit proteolytic complex that resides in the nucleus and cytoplasm and contains three subunits with different catalytic activities: β1, β2, and β5. Here, we studied how SylA targets the plant proteasome in living cells using activity-based profiling and imaging. We further developed this technology by introducing new, more selective probes and establishing procedures of noninvasive imaging in living Arabidopsis (Arabidopsis thaliana) cells. These studies showed that SylA preferentially targets β2 and β5 of the plant proteasome in vitro and in vivo. Structure-activity analysis revealed that the dipeptide tail of SylA contributes to β2 specificity and identified a nonreactive SylA derivative that proved essential for imaging experiments. Interestingly, subcellular imaging with probes based on epoxomicin and SylA showed that SylA accumulates in the nucleus of the plant cell and suggests that SylA targets the nuclear proteasome. Furthermore, subcellular fractionation studies showed that SylA labels nuclear and cytoplasmic proteasomes. The selectivity of SylA for the catalytic subunits and subcellular compartments is discussed, and the subunit selectivity is explained by crystallographic data.     

A hormone-dependent module regulating energy balance

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.     

Significance of the class II hydrophobin FgHyd5p for the life cycle of Fusarium graminearum

Hydrophobins are small secreted proteins ubiquitously found in filamentous fungi. Some hydrophobins were shown to have functions in fungal development, while others lack known function. Class II hydrophobins from Fusarium graminearum and Fusarium culmorum are characterized by formation of low stability aggregates and their solubility in organic solvents. They are economically relevant to the brewing industry because they can induce beer gushing. Since cellular functions of Hyd5p's are still unknown, we analyzed the influence of FgHyd5p on growth and morphology of F. graminearum using FgΔhyd5 knock-out mutants expressing sGFP under the control of the hyd5 promoter and compared them with the performance of the parent wild type strain. Results demonstrate that FgHyd5p does not affect the colony and hyphal morphology. FgHyd5p affects the hydrophobicity of aerial mycelia but had no obvious function in penetration of hyphae through the water air interface. The hydrophobin affects the morphology of conidia, but not their fitness. Different sources of carbon and nitrogen as well as different pH have no effect on the expression of the hyd5 gene, which was demonstrated to be expressed upon growth of F. graminearum on hydrophobic surfaces.     

Interactive Local Super-Resolution Reconstruction of Whole-Body MRI Mouse Data: A Pilot Study with Applications to Bone and Kidney Metastases

In small animal imaging studies, when the locations of the micro-structures of interest are unknown a priori, there is a simultaneous need for full-body coverage and high resolution. In MRI, additional requirements to image contrast and acquisition time will often make it impossible to acquire such images directly. Recently, a resolution enhancing post-processing technique called super-resolution reconstruction (SRR) has been demonstrated to improve visualization and localization of micro-structures in small animal MRI by combining multiple low-resolution acquisitions. However, when the field-of-view is large relative to the desired voxel size, solving the SRR problem becomes very expensive, in terms of both memory requirements and computation time. In this paper we introduce a novel local approach to SRR that aims to overcome the computational problems and allow researchers to efficiently explore both global and local characteristics in whole-body small animal MRI. The method integrates state-of-the-art image processing techniques from the areas of articulated atlas-based segmentation, planar reformation, and SRR. A proof-of-concept is provided with two case studies involving CT, BLI, and MRI data of bone and kidney tumors in a mouse model. We show that local SRR-MRI is a computationally efficient complementary imaging modality for the precise characterization of tumor metastases, and that the method provides a feasible high-resolution alternative to conventional MRI.