Differential transcript expression in the <i>ntrC</i> mutant compared to the wild type.

<p>MA plot showing the log₂ fold change in transcript expression of <i>B</i>. <i>cenocepacia</i> H111 wild type and <i>ntrC</i> mutant strains grown under nitrogen limited conditions. The top regulated genes (p-value < 0.001, absolute log₂ (Fold Change) > 1.5) are shown in color: genes with increased transcription in the <i>ntrC</i> mutant compared to the wild type are indicated in orange, down-regulated genes in green. The names of the genes of particular interest are labelled.</p

NtrC–dependent EPS production.

<p>EPS production in the wild type (1), the <i>ntrC</i> mutant (2) and the complemented strain (3) was tested on YEM plates. Three independent biological replicates were tested; the result of one is shown here.</p

NtrC-dependent control of exopolysaccharide synthesis and motility in <i>Burkholderia cenocepacia</i> H111

<div><p><i>Burkholderia cenocepacia</i> is a versatile opportunistic pathogen that survives in a wide variety of environments, which can be limited in nutrients such as nitrogen. We have previously shown that the sigma factor σ⁵⁴ is involved in the control of nitrogen assimilation and virulence in <i>B</i>. <i>cenocepacia</i> H111. In this work, we investigated the role of the σ⁵⁴ enhancer binding protein NtrC in response to nitrogen limitation and in the pathogenicity of H111. Of 95 alternative nitrogen sources tested the <i>ntrC</i> showed defects in the utilisation of nitrate, urea, L-citrulline, acetamide, DL-lactamide, allantoin and parabanic acid. RNA-Seq and phenotypic analyses of an <i>ntrC</i> mutant strain showed that NtrC positively regulates two important phenotypic traits: exopolysaccharide (EPS) production and motility. However, the <i>ntrC</i> mutant was not attenuated in <i>C</i>. <i>elegans</i> virulence.</p></div

List of the 150 genes with statistically significant differential expression, comparing the expression profile of the <i>ntrC</i> mutant with the profile of the wild type (DE-Seq analysis, p-value < 10⁻²⁰, absolute log₂(Fold Change) > 2.3).

<p>List of the 150 genes with statistically significant differential expression, comparing the expression profile of the <i>ntrC</i> mutant with the profile of the wild type (DE-Seq analysis, p-value < 10⁻²⁰, absolute log₂(Fold Change) > 2.3).</p

Validation of selected RNA-Seq results by qPCR.

<p>Validation of selected RNA-Seq results by qPCR.</p

Nitrogen sources differentially utilised by the wild-type, <i>ntrC</i> mutant and the complemented strains.

<p>Cells were grown at 37°C for 4 days with slow agitation in order to synchronize the growth. 3 independent cultures of each strain were tested with each N source. Columns and error bars indicate the average and the standard deviation of final OD₆₀₀, respectively.</p

NtrC–dependent motility.

<p>The histograms show the swarming (A) and swimming (B) motility of the <i>ntrC</i> mutant and the complemented mutant relative to the wild-type strain H111. Both assays were performed in triplicate. Significance was calculated by comparing the mutant or the complemented mutant with the wild type (* p<0.05 and ** p<0.01).</p

Biofilm formation is only slightly dependent on NtrC.

<p>Biofilm production was assessed for the wild type, <i>ntrC</i> mutant and complemented strain in 96-well plates. The columns represent the mean biofilm index generated from independent biological triplicate cultures of each strain. The error bars indicate the standard deviation. The increased biofilm formation in the complemented mutant compared to the wild type was statistically significant (*** p<0.0001). However, the slight reduction in the mutant was not statistically significant (ns).</p

<i>Nrxn</i> exon AS4 alternative splicing control is dependent on the physiological expression of T-STAR protein even though Sam68 is co-expressed.

<p>(A) Immunolocalisation of T-STAR and Sam68 proteins in the mouse hippocampus from wild type or knockout mouse brains (Abbreviations: DG - Dentate Gyrus; and AH -Ammon's Horn). The scale bar is equivalent to 20 µm). (B) Immunolocalisation in the mouse testis. Paraffin embedded adult mouse testis sections were stained with affinity purified antibodies raised against T-STAR or Sam68 (brown staining), and counterstained with haematoxylin (blue). Abbreviations: Spg –spermatogonia; Spc –spermatocyte; Rtd –round spermatid; Spd –elongating spermatid; SC –Sertoli cell. The size bar corresponds to 20 µM. (C) Levels of <i>Nrxn1</i> and <i>Nrxn3</i> AS4 alternative splice isoforms in the testes of different mouse genotypes (n = 3 mice of each genotype) measured by RT-PCR and agarose gel electrophoresis. (D) Quantification of Percentage Splicing Exclusion in the testes of different mouse genotypes using capillary gel electrophoresis (n = 3 mice of each genotype: wild type mice <i>Khdrbs3^+/+</i> (abbreviated WT) <i>Khdrbs3^+/−</i> mice (abbreviated HET) and <i>Khdrbs3^−/−</i> mice (abbreviated KO). The p values were calculated using unpaired t tests, to determine the significance of the difference between percentage splicing exclusion levels in the wild type versus either the heterozygous <i>Khdrbs^+/−</i> mice (HET); or wild type versus the homozygous <i>Khdrbs3^−/−</i> (KO) mice. The standard error of the mean is shown as an error bar.</p

T-STAR protein regulates region-specific splicing of <i>Neurexin1-3 AS4</i> in the mouse brain.

<p>(A) <i>Neurexin</i> splicing regulation in different regions of the mouse brain (B) Schematic of the different mouse brain regions used for analysis. (C–E) Percentage splicing exclusion in different regions of the mouse brain (n = 3 mice from each genotype) measured in RNA samples from wild type (column +) and knockout (column −) mice for AS4 of (C) <i>Nrxn1</i>, (D) <i>Nrxn2</i> and (E) <i>Nrxn3</i>. The error bars correspond to the standard error of the mean. Statistical significances were calculated using a two tailed unpaired t test. No splicing exclusion was observed for <i>Nrxn3</i> in the absence of T-STAR protein in any brain region.</p