A Streamlined Method to Obtain Biologically Active TcdA and TcdB Toxins from Clostridioides difficile.

The major virulence factors of Clostridioides difficile (C. difficile) are enterotoxins A (TcdA) and B (TcdB). The study of toxins is a crucial step in exploring the virulence of this pathogen. Currently, the toxin purification process is either laborious and time-consuming in C. difficile or performed in heterologous hosts. Therefore, we propose a streamlined method to obtain functional toxins in C. difficile. Two C. difficile strains were generated, each harboring a sequence encoding a His-tag at the 3' end of C. difficile 630∆erm tcdA or tcdB genes. Each toxin gene is expressed using the Ptet promoter, which is inducible by anhydro-tetracycline. The obtained purification yields were 0.28 mg and 0.1 mg per liter for rTcdA and rTcdB, respectively. In this study, we successfully developed a simple routine method that allows the production and purification of biologically active rTcdA and rTcdB toxins with similar activities compared to native toxins.

Synthesis of the NarP response regulator of nitrate respiration in Escherichia coli is regulated at multiple levels by Hfq and small RNAs.

Two-component systems (TCS) and small RNAs (sRNA) are widespread regulators that participate in the response and the adaptation of bacteria to their environments. TCSs and sRNAs mostly act at the transcriptional and post-transcriptional levels, respectively, and can be found integrated in regulatory circuits, where TCSs control sRNAs transcription and/or sRNAs post-transcriptionally regulate TCSs synthesis. In response to nitrate and nitrite, the paralogous NarQ-NarP and NarX-NarL TCSs regulate the expression of genes involved in anaerobic respiration of these alternative electron acceptors to oxygen. In addition to the previously reported repression of NarP synthesis by the SdsN137 sRNA, we show here that RprA, another Hfq-dependent sRNA, also negatively controls narP. Interestingly, the repression of narP by RprA actually relies on two independent mechanisms of control. The first is via the direct pairing of the central region of RprA to the narP translation initiation region and presumably occurs at the translation initiation level. In contrast, the second requires only the very 5' end of the narP mRNA, which is targeted, most likely indirectly, by the full-length or the shorter, processed, form of RprA. In addition, our results raise the possibility of a direct role of Hfq in narP control, further illustrating the diversity of post-transcriptional regulation mechanisms in the synthesis of TCSs.

Bacterial Small RNAs in Mixed Regulatory Networks.

Small regulatory RNAs are now recognized as key regulators of gene expression in bacteria. They accumulate under specific conditions, most often because their synthesis is directly controlled by transcriptional regulators, including but not limited to alternative sigma factors and response regulators of two-component systems. In turn, small RNAs regulate, mostly at the posttranscriptional level, expression of multiple genes, among which are genes encoding transcriptional regulators. Small RNAs are thus embedded in mixed regulatory circuits combining transcriptional and posttranscriptional controls, and whose properties are discussed here.

Mechanistic study of base-pairing small regulatory RNAs in bacteria.

In all three kingdoms of life, RNA is not only involved in the expression of genetic information, but also carries out extremely diverse cellular functions. This versatility is essentially due to the fact that RNA molecules can exploit the power of base pairing to allow them to fold into a wide variety of structures through which they can perform diverse roles, but also to selectively target and bind to other nucleic acids. This is true in particular for bacterial small regulatory RNAs that act by imperfect base-pairing with target mRNAs, and thereby control their expression through different mechanisms. Here we outline an overview of in vivo and in vitro approaches that are currently used to gain mechanistic insights into how these sRNAs control gene expression in bacteria.

Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system.

Two-component systems (TCS) and small regulatory RNAs (sRNAs) are both widespread regulators of gene expression in bacteria. TCS are in most cases transcriptional regulators. A large class of sRNAs act as post-transcriptional regulators of gene expression that modulate the translation and/or stability of target-mRNAs. Many connections have been recently unraveled between these two types of regulators, resulting in mixed regulatory circuits with poorly characterized properties. This study focuses on the negative feedback circuit that exists between the EnvZ-OmpR TCS and the OmrA/B sRNAs. We have shown that OmpR directly activates transcription from the omrA and omrB promoters, allowing production of OmrA/B sRNAs that target multiple mRNAs, including the ompR-envZ mRNA. This control of ompR-envZ by the Omr sRNAs does not affect the amount of phosphorylated OmpR, i.e. the presumably active form of the regulator. Accordingly, expression of robust OmpR targets, such as the ompC or ompF porin genes, is not affected by OmrA/B. However, we find that several OmpR targets, including OmrA/B themselves, are sensitive to changing total OmpR levels. As a result, OmrA/B limit their own synthesis. These findings unravel an additional layer of control in the expression of some OmpR targets and suggest the existence of differential regulation within the OmpR regulon.