Plasmid Curing is a Promising Approach to Improve Thermophiles for Biotechnological Applications: Perspectives in Archaea

Thermophiles are attractive as host cells for microbial processes to produce or degrade various compounds. In these applications, it is often desirable to improve the properties of thermophiles, such as their growth rate, cell density, and protein productivity, although this is rarely achieved because of the lack of general approaches. In this chapter, we describe the elimination of the pHTA426 plasmid from a moderate thermophile, Geobacillus kaustophilus HTA426, and its effects on the microbial properties. This process, called plasmid curing, was simply achieved using a DNA intercalator and confirmed by phenotypic and genotypic analyses. Of note, pHTA426 curing had beneficial effects on diverse properties, probably because of the reduced energy burden in terms of plasmid replication at high temperatures. The result suggests that plasmid curing is a simple and versatile approach for improving thermophiles. In particular, this approach may be effective for archaeal thermophiles because they grow at much higher temperatures and could have the greater energy burden on plasmid replication. Data mining has also shown that plasmids are distributed in archaeal thermophiles. This chapter provides a new tip for improving archaeal thermophiles, thereby increasing the opportunities for their use in various biotechnological applications

Electrochemical response of biased nanoelectrodes in solution

Novel approaches to DNA sequencing and detection require the measurement of electrical currents between metal probes immersed in ionic solution. Here, we experimentally demonstrate that these systems maintain large background currents with a transient response that decays very slowly in time and noise that increases with ionic concentration. Using a non-equilibrium stochastic model, we obtain an analytical expression for the ionic current that shows these results are due to a fast electrochemical reaction at the electrode surface followed by the slow formation of a diffusion layer. During the latter, ions translocate in the weak electric field generated after the initial rapid screening of the strong fields near the electrode surfaces. Our theoretical results are in very good agreement with experimental findings

DEVELOPMENT OF EQUIPMENT FOR ESTIMATING SWIMMING POWER

INTRODUCTION: In previous studies on this topic, the methods of measuring swimming power during swimming were examined. Costill et al. (1983) developed the method of evaluating swimming power by improvement of the biokinetic swim bench. It was suggested that swimming power is one of the more important components of the sprint swimming performance. However, it was difficult to apply the equipment for measuring swimming power to the swimming training, because the equipment for measuring this was complex. Therefore the purpose of this study is to develop simple equipment for estimating swimming power

Gas Phase Train in Upstream Oil & Gas Fields: PART-I Model Development

The prime contribution of this paper is to provide a large scale system (LSS) model for the gas phase operation in upstream oil and gas plants. The process model consists of the three main gas conditioning processes which exist in most upstream oil and gas processing plants; these are gas sweetening, gas dehydration, and hydrocarbon dew-pointing. The function of such a model is to provide a realistic process representation to test and verify different process control approaches, specifically those which deal with highly interactive control loops

Genomic insights into triple-negative and HER2-positive breast cancers using isogenic model systems

Introduction In general, genomic signatures of breast cancer subtypes have little or no overlap owing to the heterogeneous genetic backgrounds of study samples. Thus, obtaining a reliable signature in the context of isogenic nature of the cells has been challenging and the precise contribution of isogenic triple negative breast cancer (TNBC) versus non-TNBC remains poorly defined. Methods We established isogenic stable cell lines representing TNBC and Human Epidermal Growth Factor Receptor 2 positive (HER2+) breast cancers by introducing HER2 in TNBC cell lines MDA-MB-231 and MDA-MB-468. We examined protein level expression and functionality of the transfected receptor by treatment with an antagonist of HER2. Using microarray profiling, we obtained a comprehensive gene list of differentially expressed between TNBC and HER2+ clones. We identified and validated underlying isogenic components using qPCR and also compared results with expression data from patients with similar breast cancer subtypes. Results We identified 544 and 1087 statistically significant differentially expressed genes between isogenic TNBC and HER2+ samples in MDA-MB-231 and MDA-MB-468 backgrounds respectively and a shared signature of 49 genes. By comparing results from MDA-MB-231 and MDA-MB-468 backgrounds with two patient microarray datasets, we identified 17 and 22 common genes with same expression trend respectively. Additionally, we identified 56 and 78 genes from MDA-MB-231 and MDA-MB-468 comparisons respectively present in our published RNA-seq data. Conclusions Using our unique model system, we have identified an isogenic gene expression signature between TNBC and HER2+ breast cancer. A portion of our results was also verified in patient data samples, indicating an existence of isogenic element associated with HER2 status between genetically heterogeneous breast cancer samples. These findings may potentially contribute to the development of molecular platform that would be valuable for diagnostic and therapeutic decision for TNBC and in distinguishing it from HER2+ subtype

Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp A11-2, reveal potential for expanding its substrate selectivity

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO2) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate–FMN–enzyme complex remains unknown. Herein, we report the crystal structures of the DBT–FMN–TdsC and DBTO–FMN–TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications

Frequent Transposition of Multiple Insertion Sequences in Geobacillus kaustophilus HTA426

Geobacillus kaustophilus HTA426 is a thermophilic bacterium whose genome harbors numerous insertion sequences (IS). This study was initially conducted to generate mutant genes for thermostable T7 RNA polymerase in G. kaustophilus; however, relevant experiments unexpectedly identified that the organism transposed multiple IS elements and produced derivative cells that expressed a silent gene via transposition. The transposed elements were diverse and included members of the IS4, IS701, IS1634, and ISLre2 families. The transposition was relatively active at elevated temperatures and generated 4–9 bp of direct repeats at insertion sites. Transposition was more frequent in proliferative cells than in stationary cells but was comparable between both cells when sigX, which encodes an extra-cytoplasmic function sigma factor, was forcibly expressed. Southern blot analysis indicated that IS transposition occurred under growth inhibitory conditions by diverse stressors; however, IS transposition was not detected in cells that were cultured under growth non-inhibitory conditions. These observations suggest that G. kaustophilus enhances IS transposition via sigX-dependent stress responses when proliferative cells were prevented from active propagation. Considering Geobacillus spp. are highly adaptive bacteria that are remarkably distributed in diverse niches, it is possible that these organisms employ IS transposition for environmental adaptation via genetic diversification. Thus, this study provides new insights into adaptation strategies of Geobacillus spp. along with implications for strong codependence between mobile genetic elements and highly adaptive bacteria for stable persistence and evolutionary diversification, respectively. This is also the first report to reveal active IS elements at elevated temperatures in thermophiles and to suggest a sigma factor that governs IS transposition

30S Beam Development and X-ray Bursts

Over the past three years, we have worked on developing a well-characterized 30S radioactive beam to be used in a future experiment aiming to directly measure the 30S(alpha,p) stellar reaction rate within the Gamow window of Type I X-ray bursts. The importance of the 30S(alpha,p) reaction to X-ray bursts is discussed. Given the astrophysical motivation, the successful results of and challenges involved in the production of a low-energy 30S beam are detailed. Finally, an overview of our future plans regarding this on-going project are presented.Comment: 7 pages, 2 figures, 5th European Summer School on Experimental Nuclear Astrophysics, Santa Tecla, Sicily, September 200