The role of Cra in regulating acetate excretion and osmotic tolerance in E. coli K-12 and E. coli B at high density growth

<p>Abstract</p> <p>Background</p> <p><it>E. coli </it>B (BL21), unlike <it>E.coli </it>K-12 (JM109) is insensitive to glucose concentration and, therefore, grows faster and produces less acetate than <it>E. coli </it>K-12, especially when growing to high cell densities at high glucose concentration. By performing genomic analysis, it was demonstrated that the cause of this difference in sensitivity to the glucose concentration is the result of the differences in the central carbon metabolism activity. We hypothesized that the global transcription regulator Cra (FruR) is constitutively expressed in <it>E. coli </it>B and may be responsible for the different behaviour of the two strains. To investigate this possibility and better understand the function of Cra in the two strains, <it>cra </it>- negative <it>E. coli </it>B (BL21) and <it>E. coli </it>K-12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and real-time PCR.</p> <p>Results</p> <p>The deletion of the <it>cra </it>gene in <it>E. coli </it>B (BL21) minimally affected the growth and maximal acetate accumulation, while the deletion of the same gene in <it>E.coli </it>K-12 (JM109) caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. <it>ppsA </it>(gluconeogenesis gene), <it>aceBA </it>(the glyoxylate shunt genes) and <it>poxB </it>(the acetate producing gene) were down-regulated in both strains, while <it>acs </it>(acetate uptake gene) was down-regulated only in <it>E.coli </it>B (BL21). These transcriptional differences had little effect on acetate and pyruvate production. Additionally, it was found that the lower growth of <it>E. coli </it>K-12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producing <it>bet </it>operon (<it>betABT</it>).</p> <p>Conclusions</p> <p>The transcriptional changes caused by the deletion of <it>cra </it>gene did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone; rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that <it>cra </it>deletion caused transcription inhibition of the <it>bet </it>operon in <it>E. coli </it>K-12 (JM109) but did not affect this operon transcription in <it>E. coli </it>B (BL21). This property, together with the insensitivity to high glucose concentrations, makes this the <it>E. coli </it>B (BL21) strain more resistant to environmental changes.</p

Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

Abstract Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols. Results Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results

Analyzing the Role of Gut Microbiota on the Onset of Autoimmune Diseases Using TNF^ΔARE Murine Model

Very little is known about disease transmission via the gut microbiome. We hypothesized that certain inflammatory features could be transmitted via the gut microbiome and tested this hypothesis using an animal model of inflammatory diseases. Twelve-week-old healthy C57 Bl/6 and Germ-Free (GF) female and male mice were fecal matter transplanted (FMT) under anaerobic conditions with TNFΔARE−/+ donors exhibiting spontaneous Rheumatoid Arthritis (RA) and Inflammatory Bowel Disease (IBD) or with conventional healthy mice control donors. The gut microbiome analysis was performed using 16S rRNA sequencing amplification and bioinformatics analysis with the HIVE bioinformatics platform. Histology, immunohistochemistry, ELISA Multiplex analysis, and flow cytometry were conducted to confirm the inflammatory transmission status. We observed RA and IBD features transmitted in the GF mice cohort, with gut tissue disruption, cartilage alteration, elevated inflammatory mediators in the tissues, activation of CD4/CD8+ T cells, and colonization and transmission of the gut microbiome similar to the donors’ profile. We did not observe a change or transmission when conventional healthy mice were FMT with TNFΔARE−/+ donors, suggesting that a healthy microbiome might withstand an unhealthy transplant. These findings show the potential involvement of the gut microbiome in inflammatory diseases. We identified a cluster of bacteria playing a role in this mechanism

Additional file 1: of Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

Table S1. A summary of run metrics of the Illumina protocol and the sub-nanomolar protocol. (DOCX 24 kb

Novel reference genes in colorectal cancer identify a distinct subset of high stage tumors and their associated histologically normal colonic tissues

© 2019 The Author(s). Background: Reference genes are often interchangeably called housekeeping genes due to 1) the essential cellular functions their proteins provide and 2) their constitutive expression across a range of normal and pathophysiological conditions. However, given the proliferative drive of malignant cells, many reference genes such as beta-actin (ACTB) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) which play critical roles in cell membrane organization and glycolysis, may be dysregulated in tumors versus their corresponding normal controls Methods: Because Next Generation Sequencing (NGS) technology has several advantages over hybridization-based technologies, such as independent detection and quantitation of transcription levels, greater sensitivity, and increased dynamic range, we evaluated colorectal cancers (CRC) and their histologically normal tissue counterparts by NGS to evaluate the expression of 21 classical reference genes used as normalization standards for PCR based methods. Seventy-nine paired tissue samples of CRC and their patient matched healthy colonic tissues were subjected to NGS analysis of their mRNAs. Results: We affirmed that 17 out of 21 classical reference genes had upregulated expression in tumors compared to normal colonic epithelial tissue and dramatically so in some cases. Indeed, tumors were distinguished from normal controls in both unsupervised hierarchical clustering analyses (HCA) and principal component analyses (PCA). We then identified 42 novel potential reference genes with minimal coefficients of variation (CV) across 79 CRC tumor pairs. Though largely consistently expressed across tumors and normal control tissues, a subset of high stage tumors (HSTs) as well as some normal tissue samples (HSNs) located adjacent to these HSTs demonstrated dysregulated expression, thus identifying a subset of tumors with a potentially distinct and aggressive biological profile. Conclusion: While classical CRC reference genes were found to be differentially expressed between tumors and normal controls, novel reference genes, identified via NGS, were more consistently expressed across malignant and normal colonic tissues. Nonetheless, a subset of HST had profound dysregulation of such genes as did many of the histologically normal tissues adjacent to such HSTs, indicating that the HSTs so distinguished may have unique biological properties and that their histologically normal tissues likely harbor a small population of microscopically undetected but metabolically active tumors

Additional file 3: of Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

Table S3. Highly comparable RPKMs resulted from RNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 1126 kb

Additional file 4: of Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

Table S4. Highly comparable RPKMs resulted from MicroRNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 95 kb

Additional file 6: of Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

Table S6. Original pdf outputs from mirDeep2. Data used in Table 1 on IsomiRs (5′ modifications, 3′ modification, and nucleotide substitution) of the miR-34 family activated by p53. (DOCX 68 kb

Additional file 5: of Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

Table S5. Highly comparable RPKMs resulted from other small noncoding RNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 2013 kb

Additional file 7: of Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

Table S7. Highly similar taxonomic hierarchy and relative abundance of classification (sheet 1) and strong correlation of number of hits (sheet 2) resulted form 16S rRNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 40 kb