An evaluation of Comparative Genome Sequencing (CGS) by comparing two previously-sequenced bacterial genomes

<p>Abstract</p> <p>Background</p> <p>With the development of new technology, it has recently become practical to resequence the genome of a bacterium after experimental manipulation. It is critical though to know the accuracy of the technique used, and to establish confidence that all of the mutations were detected.</p> <p>Results</p> <p>In order to evaluate the accuracy of genome resequencing using the microarray-based Comparative Genome Sequencing service provided by Nimblegen Systems Inc., we resequenced the <it>E. coli </it>strain W3110 Kohara using MG1655 as a reference, both of which have been completely sequenced using traditional sequencing methods. CGS detected 7 of 8 small sequence differences, one large deletion, and 9 of 12 IS element insertions present in W3110, but did not detect a large chromosomal inversion. In addition, we confirmed that CGS also detected 2 SNPs, one deletion and 7 IS element insertions that are not present in the genome sequence, which we attribute to changes that occurred after the creation of the W3110 lambda clone library. The false positive rate for SNPs was one per 244 Kb of genome sequence.</p> <p>Conclusion</p> <p>CGS is an effective way to detect multiple mutations present in one bacterium relative to another, and while highly cost-effective, is prone to certain errors. Mutations occurring in repeated sequences or in sequences with a high degree of secondary structure may go undetected. It is also critical to follow up on regions of interest in which SNPs were not called because they often indicate deletions or IS element insertions.</p

Phase II Proof-of-Concept Trial of the Orexin Receptor Antagonist Filorexant (MK-6096) in Patients with Major Depressive Disorder.

BackgroundWe evaluated the orexin receptor antagonist filorexant (MK-6096) for treatment augmentation in patients with major depressive disorder.MethodsWe conducted a 6-week, double-blind, placebo-controlled, parallel-group, Phase II, proof-of-concept study. Patients with major depressive disorder (partial responders to ongoing antidepressant therapy) were randomized 1:1 to once-daily oral filorexant 10 mg or matching placebo.ResultsDue to enrollment challenges, the study was terminated early, resulting in insufficient statistical power to detect a prespecified treatment difference; of 326 patients planned, 129 (40%) were randomized and 128 took treatment. There was no statistically significant difference in the primary endpoint of change from baseline to week 6 in Montgomery Asberg Depression Rating Scale total score; the estimated treatment difference for filorexant-placebo was -0.7 (with negative values favoring filorexant) (P=.679). The most common adverse events were somnolence and suicidal ideation.ConclusionsThe interpretation of the results is limited by the enrollment, which was less than originally planned, but the available data do not suggest efficacy of orexin receptor antagonism with filorexant for the treatment of depression. (Clinical Trial Registry: clinicaltrials.gov: NCT01554176)

Ethanol and Anaerobic Conditions Reversibly Inhibit Commercial Cellulase Activity in Thermophilic Simultaneous Saccharification and Fermentation (tSSF)

A previously developed mathematical model of low solids thermophilic simultaneous saccharification and fermentation (tSSF) with Avicel was unable to predict performance at high solids using a commercial cellulase preparation (Spezyme CP) and the high ethanol yield Thermoanaerobacterium saccharolyticum strain ALK2. The observed hydrolysis proceeded more slowly than predicted at solids concentrations greater than 50 g/L Avicel. Factors responsible for this inaccuracy were investigated in this study

Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

Background: The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber \u3e 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results: The rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that \u3e 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners—Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum—exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%. Conclusions: This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading

Functional Heterologous Expression of an Engineered Full Length Cipa from Clostridium Thermocellum in Thermoanaerobacterium Saccharolyticum

Background: Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. Results:We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipAfrom C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a ΔcipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome. Conclusion: This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA . In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose

Genome-scale resources for Thermoanaerobacterium saccharolyticum

Background Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation. Results Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results. Conclusion These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum. Electronic supplementary material The online version of this article (doi:10.1186/s12918-015-0159-x) contains supplementary material, which is available to authorized users

Astrometry and geodesy with radio interferometry: experiments, models, results

Summarizes current status of radio interferometry at radio frequencies between Earth-based receivers, for astrometric and geodetic applications. Emphasizes theoretical models of VLBI observables that are required to extract results at the present accuracy levels of 1 cm and 1 nanoradian. Highlights the achievements of VLBI during the past two decades in reference frames, Earth orientation, atmospheric effects on microwave propagation, and relativity.Comment: 83 pages, 19 Postscript figures. To be published in Rev. Mod. Phys., Vol. 70, Oct. 199

Phenotype Sequencing: Identifying the Genes That Cause a Phenotype Directly from Pooled Sequencing of Independent Mutants

Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50–100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a “Phenotype Sequencing” approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only$1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110–$340