Rôle de la terminaison de la transcription Rho-dépendante dans la régulation de l'expression génique chez Bacillus subtilis

La transcription bactérienne est un processus dans lequel l'information codée dans l'ADN est transféré à l'ARN messager (ARNm). Au cours de la dernière étape de ce procédé, la terminaison de la transcription, l'ARNm est libéré et peut être utilisé pour la synthèse des protéines. Un type de terminaison de la transcription décrit chez les bactéries est la terminaison Rho-dépendante. Le rôle de Rho a été largement étudié dans le modèle à Gram négatif bactérie, Escherichia coli dans laquelle Rho est une protéine essentiel est abondant. En revanche, la connaissance de Rho chez les bactéries qui il ne sont pas essentiels et est présent en faibles quantités: par exemple Gram-positif Bacillus subtilis reste limité..Pour étudier le rôle de Rho dans le contrôle de l'expression des gènes chez B. subtilis plusieurs analyses à grande échelle ont été réalisées, y compris des tests d'interactions physiques et fonctionnelles et une analyse globale des changements observés dans l'expression des gènes en corrélation avec la production de protéines.En effet, un ensemble des Rho-spécifiques interactions physiques et fonctionnelles ont été établies. En outre, de nouveaux phénotypes de mutant dépourvu de rho ont été décrits ce qui élucider le rôle de Rho dans le contrôle des différents aspects de la physiologie cellulaire.Bacterial transcription is a process in which the information encoded in DNA is transferred to messenger RNA (mRNA). During the final step of this process, transcription termination, mRNA is released and can be used for protein synthesis. One type of transcription termination described in bacteria is Rho-dependent termination. The role of Rho has been widely investigated in model Gram-negative bacterium, Escherichia coli in which Rho is essential an abundant protein. In contrast, the knowledge about Rho inbacteria in which it is not essential and is present in low amounts, i. e. Gram-positive Bacillus subtilis remains limited.To investigate the role of Rho in control of gene expression in B. subtilis several large-scale analysis were performed. In effect, a set of Rho-specific physical and functional interactions were established. Additionally, new phenotypes of rho-null mutant were described unraveling the role of Rho in control of different aspects of cell physiology

Transcription termination factor Rho: a hub linking diverse physiological processes in bacteria.

Factor-dependent termination of transcription in bacteria relies on the activity of a specific RNA helicase, the termination factor Rho. Rho is nearly ubiquitous in bacteria, but the extent to which its physiological functions are conserved throughout the different phyla remains unknown. Most of our current knowledge concerning the mechanism of Rho's activity and its physiological roles comes from the model micro-organism Escherichia coli, where Rho is essential and involved in the control of several important biological processes. However, the rather comprehensive knowledge about the general mechanisms of action and activities of Rho based on the E. coli paradigm cannot be directly extrapolated to other bacteria. Recent studies performed in different species favour the view that Rho-dependent termination plays a significant role even in bacteria where Rho is not essential. Here, we summarize the current state of the ever-increasing knowledge about the various aspects of the physiological functions of Rho, such as limitation of deleterious foreign DNA expression, control of gene expression, suppression of pervasive transcription, prevention of R-loops and maintenance of chromosome integrity, focusing on similarities and differences of the activities of Rho in various bacterial species

Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis.

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho-null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks

Rho inactivation increases expression of KinA and KinB kinases.

<p>WT (W) and RM (r) cells containing <i>kinA</i>-SPA or <i>kinB</i>-SPA translational fusions at natural chromosomal loci were grown in LB (lanes 1–4) or sporulation-inducing DS medium (lanes 5–10) to mid-exponential (expo; OD₆₀₀ ∼ 0.5) or stationary (stat; OD₆₀₀∼ 1.5) phases and analyzed for KinA and KinB proteins using ANTI-FLAG M2 monoclonal antibodies. Equal amounts of protein were loaded onto the gel as quantified by the Bradford assay. To control equilibrium between the samples, total protein extracts from cells with <i>kinB</i>-SPA fusion were analyzed for MreB protein using anti-MreB specific antibodies.</p

Impact of Rho inactivation on swarming motility of <i>B</i>. <i>subtilis</i> cells.

<p>(A) Motility defect of the NCIB 3610 RM cells can be partially suppressed by the deletion of <i>slrR</i> and ectopic expression of <i>flhO-flhP</i> genes. Bacterial cultures were grown to an OD₆₀₀ 0.5, concentrated and spotted on the plate as described (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006909#sec019" target="_blank">Materials and methods</a>). The images were acquired after 20 hours of incubation at 37°C. Each icon represents top-grown image of centrally inoculated Petri plate (diameter 9 cm) containing LB and 0.7% of agar. Relevant genotypes are indicated on the side of each image. The repaired back to the wild type NCIB 3610 RM is denoted as <i>rho</i> wt*. The experiment was reproduced at least five times and included three biological replicas for each strain. The results from the representative experiment are presented. (B) Quantitative swarming assay of the indicated NCIB 3610 (blue lines) and isogenic NCIB 3610 RM (red lines) derivative strains. Values represent the mean of at least five experiments. (C) Impact of <i>rho</i> deletion on sense and antisense transcription of the <i>flhO-flhP</i> operon in the <i>B</i>. <i>subtilis</i> 1012 cells. Expression profiles are from the <i>B</i>. <i>subtilis</i> expression data browser (<a href="http://genome.jouy.inra.fr/cgi-bin/seb/index.py" target="_blank">http://genome.jouy.inra.fr/cgi-bin/seb/index.py</a>). Vertical bar on the top line indicates position of predicted putative terminator (shown in D). Sections show annotated genome (top) and expression profiles on the (+) and (–) strands (mid and bottom sections). Wild type (black) and RM (red) profiles are shown. (D) MFOLD [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006909#pgen.1006909.ref083" target="_blank">83</a>] predicted secondary structure (ΔG = −16, 30) within <i>flhP</i> asRNA.</p

Rho inactivation increases Spo0A phosphorylation.

<p><i>B</i>. <i>subtilis</i> BSB1 WT (blue lines) and RM (red lines) cells bearing transcription fusions of luciferase gene <i>luc</i> with the promoters of <i>spo0A</i> (A and B; solid lines), <i>tapA</i> (A; lines with squares), <i>spoIIAA</i> (C) and <i>gerE</i> (D) genes were analyzed for Luc expression during growth in biofilm-promoting MSgg medium (A) and sporulation-inducing DS medium (B, C and D) as described in Materials and Methods. Measurements were taken every 5 minutes after cells inoculation in media at optical density OD₆₀₀ ∼0.025 (time point 0). For each strain, plotted are the mean values of luminescence readings corrected for OD from four independent cultures analyzed simultaneously. In (A and B), double-lined curves depict characteristic growth kinetics of cells measured by OD 600nm. In (B), arrow indicates entry in sporulation (T0) as established in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006909#pgen.1006909.ref096" target="_blank">96</a>]. In (C) and (D), shadowed double-lined curves reproduce kinetics of <i>spo0A</i> expression established in (B) during the same experiment. The experiment was reproduced at least three times. The results from the representative experiment are presented.</p

<i>B</i>. <i>subtilis kinB</i> gene contains intragenic Rho-dependent terminator.

<p>(A and B) Schema of the experimental design used for analysis of the <i>kinB</i> putative Rho-dependent terminator. (A) Cartoon of the <i>kinB</i> expression unit and the expression profiles of <i>kinB</i> in the WT (black) and RM (red) cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006909#pgen.1006909.ref017" target="_blank">17</a>]. (B) Transcription initiation region (small red rectangle) and the 5’-terminal parts of <i>kinB</i> gene were cloned at the plasmid pGKV210 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006909#pgen.1006909.ref116" target="_blank">116</a>] upstream the promoter-less chloramphenicol-resistance gene (open arrow). The cloned fragments are delineated by the dotted lines. (C) Rho activity determines cellular resistance to chloramephenicol. <i>B</i>. <i>subtilis</i> BSB1 WT and RM cells containing pKinB-S<i>hort</i> (pKinB-S) and pKinB-L<i>ong</i> (pKinB-L) plasmids were grown to OD 0.5 and platted in sequential dilutions at the LB-plates containing or not chloramphenicol (Cm) at the indicated concentrations (μg/ml). Cm-resistant cells were scored after 24 hours of incubation at 37°C and compared to total number of viable cells. The bars represent average values from three independent experiments totally including twelve biological replicas for each strain. (D-G) Initiation rate of <i>kinB</i> translation negatively affects efficiency of Rho-dependent intragenic termination of <i>kinB</i> transcription. (D) Nucleotide sequence of the translation initiation regions (TIR) carrying native (RBSwt; [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006909#pgen.1006909.ref112" target="_blank">112</a>]) and the modified strong (RBSm+) or weak (RBSm-) ribosome binding sites. The RBS sequences are bolded and underlined. The whole modifications of TIR sequences are bolded and in italics. The <i>kinB</i> transcriptional start (+1) and ATG codon are underlined. (E, F) <i>B</i>. <i>subtilis</i> BSB1 WT cells carrying pKinB-S or pKinB-L plasmids with different <i>kinB</i> RBS (RBSwt, RBSm+ and RBSm-) were analyzed for Cm-resistance as described in (C). (E) Modifications of <i>kinB</i> RBS have no effect on Cm-resistance when plasmids do not contain transcription terminator within <i>kinB</i> (pKinB-S). (F) In the presence of <i>kinB</i> transcription terminator, the level of Cm-resistance depends on the strength of <i>kinB</i> RBS (pKinB-L). (G) The pKinB-L-RBSm- plasmid with a weak RBS determines high level of Cm-resistance after Rho inactivation in RM cells. Each experiment depicted in (E-G) included three biological replicas of each strain and was repeated at least three times. The data for WT cells with pKinB-S and pKinB-L plasmids presented in (E) and (F) are independent from (C).</p

The components of Spo0A phosphorelay differently expressed in <i>B</i>. <i>subtilis</i> WT and RM cells.

<p>The components of Spo0A phosphorelay differently expressed in <i>B</i>. <i>subtilis</i> WT and RM cells.</p

Rho inactivation accelerates sporulation of <i>B</i>. <i>subtilis</i> cells.

<p>(A) Sporulation kinetics of <i>B</i>. <i>subtilis</i> BSB1 WT and RM cells. Cells were grown in sporulation-inducing DS medium at 37°C with vigorous aeration up to OD₆₀₀ 1.5. Starting from this time-point (sporulation point T0), samples were taken from cultures each hour and analyzed for spores by heating at 75°C as described in Materials and Methods. Sporulation efficiency was estimated as proportion of viable cells in the heated and unheated cultures. Plotted are the average values and standard deviations from four independent experiments each incorporating three biological replicas of each strain. (B) Sporulation efficiency of the BSB1, PY79, NCIB 3610 and TF8A WT strains and their respective RM derivatives at sporulation point T7. Cells were grown in DS medium during seven hours after T0 and analyzed for heat resistant spores as described in (A). (C) Sporulation efficiency of the BSB1 WT, BSB1 RM strains and their respective <i>kinA</i> and <i>kinB</i> mutants. Cells were inoculated in DS medium at OD₆₀₀ 0.05, incubated at 37° during 20 hours and analyzed for spores as in (A). Totally, nine biological replicas of each strain were analyzed for (B) and twelve replicas for (C) in three independent experiments. Plotted are the average values with standard deviation error bars. (D) Kinetics of luciferase expression from <i>spoIIA-luc</i> fusion in the BSB1 WT (blue lines), BSB1 RM (red lines) and their respective <i>kinA</i> (light lines) and <i>kinB</i> (double lines) derivatives during growth in DS medium as described in Materials and Methods. For each strain, plotted are the mean values of relative luminescence readings corrected for OD from four independent cultures analyzed simultaneously. The experiment was reproduced at least three times. The results from the representative experiment are presented.</p