Confirmation of predicted transcriptional start sites (TSS) by 5′-RACE.

a<p>: Annotation according to <a href="http://www.pseudomonas.com" target="_blank">www.pseudomonas.com</a>.</p>b<p>: TSS-location as predicted by RNA-seq is <u>underlined</u>, possible TSS locations as detected by 5′-RACE are embraced in square brackets. The ambiguity results from poly-A tailing (see text).</p>c<p>: Numbers correspond to the position range of the putative TSS nucleotides as marked by square brackets in the sequence context.</p>d<p>: RNA-seq data showed two adjacent peaks upstream of <i>mexR</i>. The smaller one (−44) was not confirmed by 5′-RACE, probably due to its low abundance.</p

Comparison of differential regulation among different studies.

<p>This qualitative ‘heatmap’ compares the differential expression of selected genes and gene clusters in B-48 with similar studies that have been published previously. In all cases, the expression of a biofilm culture was compared to a planktonic culture. The studies included in this analysis are Whiteley <i>et al.... </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031092#pone.0031092-Whiteley1" target="_blank">[1]</a>, Waite <i>et al.... </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031092#pone.0031092-Waite1" target="_blank">[21]</a>, Hentzer <i>et al.... </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031092#pone.0031092-Hentzer1" target="_blank">[4]</a> and Mikkelsen <i>et al.... </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031092#pone.0031092-Mikkelsen1" target="_blank">[20]</a>. The method abbreviations are CS – chemostat (continuous culture), 96-well – static 96-well plate cultures, agar – nitrocellulose filter on agar surface, tube – silicon tubes, array – DNA microarray analysis, seq – sequencing of cDNA.</p

Detection of transcriptional start sites.

<p>A) Transcriptional start sites (TSS) were categorized by their position relative to annotated genes as follows: promoter (P), up to 500 bp upstream of an annotated gene on the same strand, intragenic (I), within an annotated gene on the same strand, antisense (A), within an annotated gene on the opposite strand, orphan (O), neither ‘intragenic’ nor ‘promoter’, which means no association to any annotated gene. B) Venn diagram depicting the abundance of the different categories among 1024 TSS that were independently detected for at least two different conditions. C) Distribution of TSS in promoter regions of coding genes (class ‘P’), indicating the length of the untranslated regions (UTRs) at the 5′ end of mRNAs. The peak of the distribution is located around the −25 position. The dashed line marks the −10 nt position, which indicates the 43 leaderless transcripts (red bars) with 5′ UTRs of less than 10 nt length.</p

Gene expression under planktonic and biofilm growth conditions.

<p>A) Venn diagram showing the genes that were differentially expressed (−2≤log₂ fold change ≤2; P≤0.001) in planktonic stationary phase cultures (P-12) and biofilms growing for 24 or 48 h (B-24, B-48) as compared to late exponential phase planktonic cultures (P-4). B) Principle component analysis of absolute gene expression. The first principle component (PC 1) accounted for 58% and PC 2 for 25% of the total variation in the dataset. Symbols indicate two biological replicates of late exponential phase planktonic cultures (P-4, •), stationary phase planktonic cultures (P-12, ○), 24 h old biofilms (B-24, ▴) and 48 h old biofilms (B-48, ▵), respectively. C) Cluster analysis of the normalized gene expression for genes that were differentially regulated in P-12, B-24 or B-48 as compared to P-4. Clusters have been labeled ‘B’ for ‘biofilm specific’ (blue background), if gene expression was consistently different between the biofilm cultures (B-24, B-48) and planktonic cultures (P-4, P-12). A comprehensive list of absolute and differential gene expression for all genes included in this figure is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031092#pone.0031092.s003" target="_blank">Table S2</a>. A larger version of this heatmap including gene labels can be viewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031092#pone.0031092.s001" target="_blank">Figure S1</a>.</p

Small RNAs that were differentially expressed in PA14 biofilms.

a<p>: annotation taken from Sonnleitner et al.</p>b<p>: relative to P-4, only at least +/− 1 log2-fold regulated genes with a P-value < = 0.001 are shown.</p

Profiles displaying the genome coverage by sequence reads.

<p>A) Profiles showing the coverage of the plus (red) and minus (green) strand of the <i>P. aeruginosa</i> PA14 genome visualized using the Artemis genome browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031092#pone.0031092-Rutherford1" target="_blank">[58]</a>. The upper profile represents a <i>pileup</i> of the sequence reads counting the number of reads overlapping each position of the chromosome. In the lower <i>sinister</i> profile, only left end (5′-) positions of sequence reads were counted. The genome annotation is shown below the profiles with numbers indicating the position on the chromosome and white boxes indicating genes on the plus strand (upper row) and the minus strand (lower row), respectively. The genes within the displayed region are <i>lasI</i> (PA14_45940), <i>rsaL</i> (PA14_45950) and <i>lasR</i> (PA14_45960). B) Proposed model for the enrichment of transcripts ends: mRNA is isolated (1.), randomly fragmented (2.) and the first 30 nucleotides of the left ends of the fragments are sequenced on an Illumina GenomeAnalyzer (3.). Since the fragmentation of each mRNA produces identical 5′-ends and all other fragments are random, mapping leads to a stack of sequence reads at the mRNA 5′-end (4.) that can be used to annotate the TSS (5.).</p

Alternative transcriptional start sites of the PQS operon.

<p>A) Model of the PQS biosynthetic operon (<i>pqsABCDE</i>) with the three alternative TSS indicated by arrows. The TSS are named by the next downstream gene with subscripts indicating the TSS position relative to the start of the associated gene. B) <i>Pileup</i> profile of the PQS operon depicting the total amount of read counts generated in this study that map to the PQS operon. The enriched TSS loci are indicated by black triangles.</p

dLint-1 is a novel dL(3)mbt–interacting protein.

<p>(A) Nuclear extract from Kc cells was fractionated over a Superose 6 column. Fractions were analyzed by Western blot using the antibodies indicated. Fraction numbers and molecular mass standards are denoted on top. Input: 5% of extract loaded. (B) Nuclear extracts from control S2 cells (mock, lanes 1 and 3) and S2 cells stably expressing FLAG-dL(3)mbt (lanes 2 and 4) were subjected to FLAG affinity purification, elution with FLAG peptide, SDS-PAGE and silver staining (lanes 3 and 4). Input: 2 µg of nuclear extracts (lanes 1 and 2). The positions of FLAG-dL(3)mbt and dLint-1 are indicated on the right. (C) <i>In vitro</i> translated, ³⁵S-labeled dLint-1 (upper panel) or luciferase (lower panel) were incubated with FLAG beads (beads, lane 3) or beads loaded with FLAG-dL(3)mbt (lane 2). Bound proteins were separated by SDS-PAGE and detected by autoradiography. Lane 1: 1% input. (D) Sf9 cells were infected with baculoviruses expressing dL(3)mbt-FLAG or dLint-1 as indicated on top. Extracts were immunoprecipitated and subjected to Western blot using FLAG and dLint-1 #2 antibodies (lanes 2, 4 and 6). Lanes 1, 3 and 5: 5% input. (E) Nuclear extracts from control S2 cells (mock, lanes 1 and 3) and S2 cells stably expressing dLint-1-FLAG (lanes 2 and 4) were subjected to FLAG affinity purification, elution with FLAG peptide, SDS-PAGE and silver staining (lanes 3 and 4). Input: 2 µg of nuclear extracts (lanes 1 and 2). The position of dLint-1-FLAG and copurifying proteins are indicated on the right. * denotes that eIF-4B was also recovered from the control and is considered to be a contaminant. Note that dCoREST and dLsd1 have the same molecular weight and comigrate. (F) Nuclear extracts from control S2 cells (mock, lanes 1 and 2) and S2 cells stably expressing FLAG-dLint-1 (lanes 3 and 4) were precipitated with FLAG antibody and analyzed by Western blot as indicated (lanes 2 and 4). dPR-Set7 served as a negative control. Lanes 1 and 3: 5% input.</p

Purification of the LINT complex.

<p>(A) Nuclear extracts of Kc cells treated with dsRNA directed against EGFP (control, lanes 1 and 3) and dLint-1 (lanes 2 and 4) were subjected to Western blot using dLint-1 antibody #1 (left upper panel), dLint-1 antibody #2 (right upper panel) and tubulin antibody (lower panels). (B) Nuclear extracts from Kc cells were precipitated with protein G beads (beads control, lane 3) and beads loaded with dLint-1 #1 antibody (lane 2) and analyzed by Western blot as indicated (lanes 2 and 3). dMi-2 served as a negative control. Lane 1: 5% input; lane 4 contains dLint-1 antibody (antibody control). * denotes a polypeptide that unspecifically crossreacts with the dLsd1 antibody (compare lanes 2 and 4). (C) Nuclear extract from Kc cells was fractionated over a Superose 6 column. Fractions were analyzed by Western blot. Fraction numbers and molecular mass standards are denoted on top. Input: 5% of extract loaded. (D) Kc nuclear extract was separated by sequential ion exchange chromatography over Q-Sepharose and MonoQ columns. MonoQ fractions were analyzed by Western blot as indicated. Fraction numbers are denoted on top. (E) Extract from third instar larval brains was precipitated with protein G beads (beads control, lane 3) and beads loaded with dLint-1 antibody (lane 2) and analyzed by Western blot as indicated (lanes 2 and 3). Lane 1: 5% input; lane 4 contains dLint-1 antibody (antibody control). * denotes a polypeptide that unspecifically crossreacts with the dLsd1 antibody (compare lanes 2 and 4).</p

Promoter recruitment of LINT subunits results in transcriptional repression.

<p>(A) A <i>Firefly</i> luciferase reporter construct (schematic representation on top) was transiently cotransfected into RNAi treated Kc cells along with a <i>Renilla</i> luciferase reporter and varying amounts of expression vectors for LexA or dL(3)mbt-/dLint-1-LexA fusion proteins as indicated. Repressor activities of dL(3)mbt-LexA and dLint-1-LexA are presented as -fold repression normalized against activities measured for LexA expression alone. (B) Kc cells were treated with no dsRNA (mock) or dsRNA against EGFP, dL(3)mbt, dLint-1, dCoREST, dLsd1, G9a and Pc. Cells were then cotransfected with reporter and expression vectors as in (A).</p