RNA-Seq-Based Analysis of Cold Shock Response in <i>Thermoanaerobacter tengcongensis</i>, a Bacterium Harboring a Single Cold Shock Protein Encoding Gene

<div><p>Background</p><p>Although cold shock responses and the roles of cold shock proteins in microorganisms containing multiple cold shock protein genes have been well characterized, related studies on bacteria possessing a single cold shock protein gene have not been reported. <i>Thermoanaerobacter tengcongensis</i> MB4, a thermophile harboring only one known cold shock protein gene (<i>TtescpC</i>), can survive from 50° to 80°C, but has poor natural competence under cold shock at 50°C. We therefore examined cold shock responses and their effect on natural competence in this bacterium.</p><p>Results</p><p>The transcriptomes of <i>T. tengcongensis</i> before and after cold shock were analyzed by RNA-seq and over 1200 differentially expressed genes were successfully identified. These genes were involved in a wide range of biological processes, including modulation of DNA replication, recombination, and repair; energy metabolism; production of cold shock protein; synthesis of branched amino acids and branched-chain fatty acids; and sporulation. RNA-seq analysis also suggested that <i>T. tengcongensis</i> initiates cell wall and membrane remodeling processes, flagellar assembly, and sporulation in response to low temperature. Expression profiles of <i>TtecspC</i> and failed attempts to produce a <i>TtecspC</i> knockout strain confirmed the essential role of <i>Tte</i>CspC in the cold shock response, and also suggested a role of this protein in survival at optimum growth temperature. Repression of genes encoding ComEA and ComEC and low energy metabolism levels in cold-shocked cells are the likely basis of poor natural competence at low temperature.</p><p>Conclusion</p><p>Our study demonstrated changes in global gene expression under cold shock and identified several candidate genes related to cold shock in <i>T. tengcongensis</i>. At the same time, the relationship between cold shock response and poor natural competence at low temperature was preliminarily elucidated. These findings provide a foundation for future studies on genetic and molecular mechanisms associated with cold shock and acclimation at low temperature.</p></div

GO analysis of differentially expressed genes in <i>T. tengcongensis</i> with and without cold shock.

<p>GO analysis of differentially expressed genes in <i>T. tengcongensis</i> with and without cold shock.</p

Comparison of gene expression between biological samples with and without cold shock.

<p>Scatterplot matrix comparison of gene expression in two biological replicates of TTE75 (A) and TTECS (B). C. Change level of global differential expression genes between TTECS and TTE75.</p

Summary of Illumina RNA-seq data.

<p>*Clean data were obtained from raw data by removing reads containing adapter, ploy-N and low quality reads.</p

Validation of differentially expressed genes by real-time PCR.

<p>Comparison of RNA-seq and real-time PCR measures of changes in expression of <i>eutD</i> (encoding phosphotransacetylase), <i>rpmE</i> (50S ribosomal protein L31), <i>acpP</i> (acyl carrier protein), <i>tte0510</i> (hypothetical protein), <i>tte2654</i> (hypothetical protein), <i>tte0106</i> (hypothetical protein), <i>dnaA</i> (chromosome replication initiator DnaA), <i>tte1002</i> (hypothetical protein), <i>pspf4</i> (a transcriptional regulator), and <i>cspC</i> (cold shock protein). All data are shown as means ± SD (n = 3).</p

KEGG pathway enrichment analysis of differentially expressed genes between TTCS and TTE75.

<p>KEGG pathway enrichment analysis of differentially expressed genes between TTCS and TTE75.</p

Top ten up- and downregulated <i>T. tengcongensis</i> genes after cold shock at 50°C for 1 h.

<p>↑: up-regulated;</p><p>↓: down-regulated;</p><p>* novel transcript detected by RNA-Seq.</p

Synthesis of Polysubstituted Furans via Copper-Mediated Annulation of Alkyl Ketones with α,β-Unsaturated Carboxylic Acids

A novel copper-mediated annulation of alkyl ketones with α,β-unsaturated carboxylic acids has been accomplished. This reaction provides a facile and regio-defined method for the synthesis of 2,3,5-trisubstituted furans from simple chemical reagents

Free-Amine Directed Arylation of Biaryl-2-amines with Aryl Iodides by Palladium Catalysis

A new palladium-catalyzed free-amine directed arylation of C(sp²)–H bonds in the presence of AgOAc and TFA is described. Biaryl-2-amines react with various aryl iodides to give the corresponding mono- or diarylated products with exclusive regioselectivity

Palladium-Catalyzed Direct C-3 Alkynylation of Indolizines with (2,2-Dibromovinyl)arenes

<div><p></p><p>The direct C-3 alkynylation of indolizines with (2,2-dibromovinyl)arenes in the presence of palladium catalyst has been developed. This novel protocol showed wide substrate scope with respect to both indolizines and dibromoalkenes. Also this method was characterized with high efficiency and good functional group tolerance.</p> </div