Horizontally acquired AT-rich genes in Escherichia coli cause toxicity by sequestering RNA polymerase

Horizontal gene transfer permits rapid dissemination of genetic elements between individuals in bacterial populations. Transmitted DNA sequences may encode favourable traits. However, if the acquired DNA has an atypical base composition, it can reduce host fitness. Consequently, bacteria have evolved strategies to minimize the harmful effects of foreign genes. Most notably, xenogeneic silencing proteins bind incoming DNA that has a higher AT content than the host genome. An enduring question has been why such sequences are deleterious. Here, we showed that the toxicity of AT-rich DNA in Escherichia coli frequently results from constitutive transcription initiation within the coding regions of genes. Left unchecked, this causes titration of RNA polymerase and a global downshift in host gene expression. Accordingly, a mutation in RNA polymerase that diminished the impact of AT-rich DNA on host fitness reduced transcription from constitutive, but not activator-dependent, promoters

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12

The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli. The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS (“sensitive” genes), and genes whose expression changes very little with the production of a little RpoS (“insensitive”). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS

A Self-Splicing Group I Intron in DNA Polymerase Genes of T7-Like Bacteriophages

Group I introns are inserted into genes of a wide variety of bacteriophages of gram-positive bacteria. However, among the phages of enteric and other gram-negative proteobacteria, introns have been encountered only in phage T4 and several of its close relatives. Here we report the insertion of a self-splicing group I intron in the coding sequence of the DNA polymerase genes of ΦI and W31, phages that are closely related to T7. The introns belong to subgroup IA2 and both contain an open reading frame, inserted into structural element P6a, encoding a protein belonging to the HNH family of homing endonucleases. The introns splice efficiently in vivo and self-splice in vitro under mild conditions of ionic strength and temperature. We conclude that there is no barrier for maintenance of group I introns in phages of proteobacteria

Comprehensive Mapping of the <i>Escherichia coli</i> Flagellar Regulatory Network

<div><p>Flagellar synthesis is a highly regulated process in all motile bacteria. In <i>Escherichia coli</i> and related species, the transcription factor FlhDC is the master regulator of a multi-tiered transcription network. FlhDC activates transcription of a number of genes, including some flagellar genes and the gene encoding the alternative Sigma factor FliA. Genes whose expression is required late in flagellar assembly are primarily transcribed by FliA, imparting temporal regulation of transcription and coupling expression to flagellar assembly. In this study, we use ChIP-seq and RNA-seq to comprehensively map the <i>E. coli</i> FlhDC and FliA regulons. We define a surprisingly restricted FlhDC regulon, including two novel regulated targets and two binding sites not associated with detectable regulation of surrounding genes. In contrast, we greatly expand the known FliA regulon. Surprisingly, 30 of the 52 FliA binding sites are located inside genes. Two of these intragenic promoters are associated with detectable noncoding RNAs, while the others either produce highly unstable RNAs or are inactive under these conditions. Together, our data redefine the <i>E. coli</i> flagellar regulatory network, and provide new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process.</p></div

Genome-wide FlhDC-dependent gene expression.

<p>Relative expression of all genes in motile MG1655 versus relative expression in Δ<i>flhDC</i>. Gene expression values represent normalized expression values calculated by Rockhopper. Values on the y-axis represent the average of normalized expression values in the Δ<i>flhD</i> and Δ<i>flhC</i> strains. Color-coding indicates which genes are associated with FlhDC (blue) or FliA (green) binding. Genes not associated with FlhDC of FliA binding are color-coded according to whether they are significantly regulated (Rockhopper, q-value≤0.01) and changed at least 2-fold (black) or not (grey).</p

FlhDC binding sites and expression of associated genes.

1<p>Normalized gene expression values generated by Rockhopper. Expression values are for first gene in operon. Values in parentheses correspond with gene name in parentheses.</p>2<p>Peak centers represent an average of peak centers determined for FlhD and FlhC. Numbers represent genome coordinates relative to NC_000913.2.</p>3<p>Fold Above Threshold (FAT).</p>4<p>Gene(s) adjacent to binding site. Parentheses indicate an intragenic binding site.</p>5<p>Asterisks indicate significant differential expression (as defined in Methods) between motile MG1655 and indicated deletion strain.</p><p>FlhDC binding sites and expression of associated genes.</p

FliA binding and regulation at known and novel targets.

<p>(A–F) Mapped reads from ChIP-seq and RNA-seq experiments. Genes and operons of interest are boxed (dotted black line) and labelled. Within each panel, both lanes of ChIP-seq data are scaled equivalently, and all three lanes of RNA-seq are scaled equivalently. Relative scales are indicated below each panel. Dotted gray lines indicate that mapped reads exceed the scale shown. (G) Gene expression, relative to <i>mreB</i>, measured by RT-PCR. Gene names are indicated on the x-axis and strain are indicated in the legend (n = 4–7). Statistical comparisons of were performed using two-sample T tests between the indicated groups: * <i>p</i><0.05, ** <i>p</i><0.01 (two-tailed).</p