Model of pilus regulation by an attenuation-like mechanism.

Abstract

<p>(A) <i>S. gallolyticus</i> UCN34 WT displays a heterogeneous <i>pil1</i> expression and consists of two main subpopulations, a majority of low piliated cells (Pil1<sub>low</sub>, 70%) and a minority of hyper piliated cells (Pil1<sub>high</sub>, 30%). The Pil1<sub>low</sub> cells, characterized by a basal expression of <i>pil1</i>, possess a regulatory leader peptide-encoding gene with 22 GCAGA repeats that ends 39 bp upstream the hairpin transcription terminator. In-frame addition/deletion of GCAGA did not modify the distance between the leader peptide stop codon and the terminator. In this case, most transcripts initiated at P<i>pil1</i> promoter end at this premature terminator and the observed low expression of <i>pil1</i> probably occurs by readthrough transcription. The Pil1<sub>high</sub> cells, characterized by a strong expression of <i>pil1</i>, displayed out-of-frame addition/deletion of repeats resulting in the synthesis of longer regulatory peptides whose stop codon are located within or dowstream of the hairpin terminator. Translation of these long regulatory leader peptides enhances <i>pil1</i> genes transcription by preventing the formation of the transcription terminator. (B) Flow cytometry analysis of UCN34 WT and isogenic mutant derivatives (Δ<i>term</i>, ATG*, and 3 STOPs) labeled with anti-PilB pAb. The WT and mutant profiles are depicted by gray area and black lines, respectively. Note that, as expected, the UCN34 and “back to the WT” profiles, depicted by dotted lines, are almost entirely superimposable. (C) Immunolabeling screening of the Pil1+<sub>var23</sub> and Δ<i>term</i> strains with anti-PilB pAb. As opposed to the Pil1+<sub>var23</sub> variant, the Δ<i>term</i> mutant is homogeneous and only generates Pil1+ colonies. This is the consequence of the deletion of the transcription terminator that blocks this strain in the Pil1<sub>high</sub> configuration. (D) Quantitative RT-PCR were performed on RNAs extracted from WT UCN34 and mutant derivatives using pilB primers. The expression levels were normalized using 16S rRNA as an internal standard and are indicated as the n-fold change with respect to WT UCN34, expressed as means and standard deviations of at least three independent experiments with four technical replicates. Asterisks represent P values (** <i>P</i><0.05) evaluated using a Student <i>t</i> test.</p

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