Homologues of epigenetic pyrimidines: 5-alkyl-, 5-hydroxyalkyl and 5-acyluracil and -cytosine nucleotides: synthesis, enzymatic incorporation into DNA and effect on transcription with bacterial RNA polymerase.

Homologues of natural epigenetic pyrimidine nucleosides and nucleotides were designed and synthesized. They included 5-ethyl-, 5-propyl-, 5-(1-hydroxyethyl)-, 5-(1-hydroxypropyl)- and 5-acetyl- and 5-propionylcytosine and -uracil 2'-deoxyribonucleosides and their corresponding 5'-O-triphosphates (dNXTPs). The epimers of 5-(1-hydroxyethyl)- and 5-(1-hydroxypropyl)pyrimidine nucleosides were separated and their absolute configuration was determined by a combination of X-ray and NMR analysis. The modified dNXTPs were used as substrates for PCR synthesis of modified DNA templates used for the study of transcription with bacterial RNA polymerase. Fundamental differences in transcription efficiency were observed, depending on the various modifications. The most notable effects included pronounced stimulation of transcription from 5-ethyluracil-bearing templates (200% transcription yield compared to natural thymine) and an enhancing effect of 5-acetylcytosine versus inhibiting effect of 5-acetyluracil. In summary, these results reveal that RNA polymerase copes with dramatically altered DNA structure and suggest that these nucleobases could potentially play roles as artificial epigenetic DNA nucleobases.

Glucosylated 5-Hydroxymethylpyrimidines as Epigenetic DNA Bases Regulating Transcription and Restriction Cleavage.

5-(ß-d-Glucopyranosyloxymethyl)-2'-deoxyuridine and -cytidine 5'-O-triphosphates were prepared and used for polymerase-mediated (primer extension or PCR) synthesis of DNA containing glucosylated 5-hydroxymethyluracil (5hmU) or 5-hydroxymethyluracil (5hmC). The presence of any glucosylated pyrimidines fully protected DNA from cleavage by type II restriction endonucleases. On the other hand, while the presence of glucosylated 5hmU completely inhibited transcription by bacterial (Escherichia coli) RNA polymerase, the DNA containing the corresponding glucosylated 5hmC allowed a similar level of transcription as natural DNA. This suggests different roles of these hypermodified bases in the epigenetic regulation of transcription in bacteriophages or kinetoplastid parasites. Consequently, enzymatic glucosylation of 5hmC-containing DNA can be used for tuning of transcription activity.

Kinetic Modeling and Meta-Analysis of the Bacillus subtilis SigB Regulon during Spore Germination and Outgrowth.

The exponential increase in the number of conducted studies combined with the development of sequencing methods have led to an enormous accumulation of partially processed experimental data in the past two decades. Here, we present an approach using literature-mined data complemented with gene expression kinetic modeling and promoter sequence analysis. This approach allowed us to identify the regulon of Bacillus subtilis sigma factor SigB of RNA polymerase (RNAP) specifically expressed during germination and outgrowth. SigB is critical for the cell's response to general stress but is also expressed during spore germination and outgrowth, and this specific regulon is not known. This approach allowed us to (i) define a subset of the known SigB regulon controlled by SigB specifically during spore germination and outgrowth, (ii) identify the influence of the promoter sequence binding motif organization on the expression of the SigB-regulated genes, and (iii) suggest additional sigma factors co-controlling other SigB-dependent genes. Experiments then validated promoter sequence characteristics necessary for direct RNAP-SigB binding. In summary, this work documents the potential of computational approaches to unravel new information even for a well-studied system; moreover, the study specifically identifies the subset of the SigB regulon, which is activated during germination and outgrowth.

Photocaged 5-(Hydroxymethyl)pyrimidine Nucleoside Phosphoramidites for Specific Photoactivatable Epigenetic Labeling of DNA.

5-Hydroxymethylcytosine and uracil are epigenetic nucleobases, but their biological roles are still unclear. We present the synthesis of 2-nitrobenzyl photocaged 5-hydroxymethyl-2'-deoxycytidine and uridine 3'-O-phosphoramidites and their use in automated solid-phase synthesis of oligonucleotides (ONs) modified at specific positions. The ONs were used as primers for PCR to construct DNA templates modified in the promoter region that allowed switching of transcription through photochemical uncaging.

Correction to: Reprogramming of gene expression in Escherichia coli cultured on pyruvate versus glucose.

The article Reprogramming of.

Reprogramming of gene expression in Escherichia coli cultured on pyruvate versus glucose.

Previous studies revealed important roles of small RNAs (sRNAs) in regulation of bacterial metabolism, stress responses and virulence. However, only a minor fraction of sRNAs is well characterized with respect to the spectra of their targets, conditional expression profiles and actual mechanisms they use to regulate gene expression to control particular biological pathways. To learn more about the specific contribution of sRNAs to the global regulatory network controlling the Escherichia coli central carbon metabolism (CCM), we employed microarray analysis and compared transcriptome profiles of E. coli cells grown on two alternative minimal media supplemented with either pyruvate or glucose, respectively. Microarray analysis revealed that utilization of these alternative carbon sources led to profound differences in gene expression affecting all major gene clusters associated with CCM as well as expression of several known (CyaR, RyhB, GcvB and RyeA) and putative (C0652) sRNAs. To assess the impact of transcriptional reprogramming of gene expression on E. coli protein abundance, we also employed two-dimensional protein gel electrophoresis. Our experimental data made it possible to determine the major pathways for pyruvate assimilation when it is used as a sole carbon source and reveal the impact of other key processes (i.e., energy production, molecular transport and cell resistance to stress) associated with the CCM in E. coli. Moreover, some of these processes were apparently controlled by GcvB, RyhB and CyaR at the post-transcriptional level, thus indicating the complexity and interconnection of the regulatory networks that control CCM in bacteria.

A new custom microarray for sRNA profiling in Escherichia coli.

Bacterial small RNAs (sRNAs) play essential roles in the post-transcriptional control of gene expression. To improve their detection by conventional microarrays, we designed a custom microarray containing a group of probes targeting known and some putative Escherichia coli sRNAs. To assess its potential in detection of sRNAs, RNA profiling experiments were performed with total RNA extracted from E. coli MG1655 cells exponentially grown in rich (Luria-Bertani) and minimal (M9/glucose) media. We found that many sRNAs could yield reasonably strong and statistically significant signals corresponding to nearly all sRNAs annotated in the EcoCyc database. Besides differential expression of two sRNAs (GcvB and RydB), expression of other sRNAs was less affected by the composition of the growth media. Other examples of the differentially expressed sRNAs were revealed by comparing gene expression of the wild-type strain and its isogenic mutant lacking functional poly(A) polymerase I (pcnB). Further, northern blot analysis was employed to validate these data and to assess the existence of new putative sRNAs. Our results suggest that the use of custom microarrays with improved capacities for detection of sRNAs can offer an attractive opportunity for efficient gene expression profiling of sRNAs and their target mRNAs at the whole transcriptome level.

A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing.

Streptomyces coelicolor is a model for studying bacteria renowned as the foremost source of natural products used clinically. Post-genomic studies have revealed complex patterns of gene expression and links to growth, morphological development and individual genes. However, the underlying regulation remains largely obscure, but undoubtedly involves steps after transcription initiation. Here we identify sites involved in RNA processing and degradation as well as transcription within a nucleotide-resolution map of the transcriptional landscape. This was achieved by combining RNA-sequencing approaches suited to the analysis of GC-rich organisms. Escherichia coli was analysed in parallel to validate the methodology and allow comparison. Previously, sites of RNA processing and degradation had not been mapped on a transcriptome-wide scale for E. coli. Through examples, we show the value of our approach and data sets. This includes the identification of new layers of transcriptional complexity associated with several key regulators of secondary metabolism and morphological development in S. coelicolor and the identification of host-encoded leaderless mRNA and rRNA processing associated with the generation of specialized ribosomes in E. coli. New regulatory small RNAs were identified for both organisms. Overall the results illustrate the diversity in mechanisms used by different bacterial groups to facilitate and regulate gene expression.