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

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

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

Genome-wide binding of FliA.

<p>(A) Genome-wide binding of FliA (green) determined by ChIP-seq. Gray boxes represent gene in the MG1655 genome. (B) ChIP-qPCR enrichment at one well-known (<i>fliC</i>) and 13 novel FliA binding sites (n = 3, * <i>p</i><0.05, ** <i>p</i><0.01). Gene names in parentheses indicate that the binding site occurs within that gene. (C) FliA motif derived in this study (all binding sites). (D) Motif derived from binding sites with FAT scores ≤4. Motifs in C and D were generated using MEME <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004649#pgen.1004649-Bailey1" target="_blank">[52]</a>. (E) Distribution of motifs relative to ChIP-seq peak centers. Motifs cluster ∼25 nt upstream of the peak center, relative to the orientation of the motif.</p

FhDC binding and regulation at known and novel targets.

<p>(A–E) 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. (F & 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><0.01 (two-tailed).</p

Updated flagellar transciption network and localization of dual-regulated targets.

<p>(A) The transcription network map has been updated to accurately depict dual-regulated targets and to incorporated novel regulon members. Ovals at top left represent positive (green) and negative (red) regulation of <i>flhDC</i> transcription. Blue arrows indicate translation of gene products. Purple ovals represent FlhDC and purple arrows indicate FlhDC regulatory interactions. Green cresents represent FliA and green arrows represent FliA regulatory interactions. Dotted arrows indicate potential regulation of genes associated with binding sites but without detectable regulation in this study. Target genes/operons are boxed according to their regulatory input: FlhDC only (green), FliA only (green), and dual FlhDC and FliA (orange). (B) Localization and function of flagellar gene products, color-coded by regulatory input as described for panel B. Gene products not physically associated with the flagellum are omitted.</p

FlhDC and FliA binding and regulation at dual regulated targets.

<p>(A–D) 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. (E) 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). Due to the high number of potential comparisons, statistical analysis is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004649#pgen.1004649.s009" target="_blank">Table S2</a>.</p

FliA promoters 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 and motif centers refer to genome coordinates relative to NC_000913.2.</p>3<p>Peak centers and motif centers in italics are likely false positives, based on the location of the motif relative to the peak center.</p>4<p>Fold Above Threshold (FAT).</p>5<p>Gene(s) adjacent to binding site. Parentheses indicate an intragenic binding site, while those that are underlined are in the sense orientation and those not underlined are in the antisense orientation.</p>6<p>Asterisks indicate significant differential expression (as defined in Methods) between motile MG1655 and Δ<i>fliA</i>.</p><p>FliA promoters and expression of associated genes.</p

Genome-wide binding of FlhD and FlhC.

<p>(A) Genome-wide binding of FlhD (blue) and FlhC (purple) determined by ChIP-seq. Gray boxes represent genes in the MG1655 genome. (B) ChIP-qPCR enrichment at one well-known (<i>fliA</i>) and 5 novel FlhDC binding sites (n = 3). ChIP enrichment in tagged strains was compared to untagged control using two-sample T tests: * <i>p</i><0.05, ** <i>p</i><0.01 (one-tailed). (C) FlhDC motif derived in this study, using BioProspector <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004649#pgen.1004649-Liu3" target="_blank">[49]</a> (score = 1.49) and visualized by inputing aligned sequenced into WebLogo <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004649#pgen.1004649-Crooks1" target="_blank">[76]</a>.</p