Regulatory cascades in Escherichia coli : the role of small RNAs and two-component systems to regulate membrane composition and magnesium homeostasis – Sydécorna

We use mostly microbiology and molecular genetics approaches.

We have shown that the synthesis of several transcriptional regulators is modulated at the post-transcriptional level by small regulatory RNAs. These multiple layers of regulation link for instance amino-acid metabolism, membrane homeostasis and the central two-component system PhoQ/PhoP. It is crucial to understand those controls that can affect essential functions such as bacterial virulence for instance.

It would be of primary interest to understand how the connections between different regulatory networks affect the functions encoded by the downstream genes, such as magnesium homeostasis. This ion has a key-role in cellular physiology, as it intervenes in the structure of nucleic acids and their interaction with other molecules.

Our data were presented in several international conferences, and have been submitted for publication in PLoS Gentics.

A major challenge faced by bacteria is the necessity to adapt rapidly to ever-changing environments, which partly relies on their ability to regulate gene expression as a function of the environmental conditions. This amazing capacity to adapt to a great variety of growth conditions is mainly due to a limited number of regulatory modes. Among them, we find the two component systems (TCS) that regulate the transcription of specific-target genes in response to environmental changes. Another regulatory mode is illustrated by the sigma-dependent cascades responding to diverse stresses such as temperature shifts or envelope stress. In this case, specific sigma factors replace the house-keeping sigma factor and activate transcription of specific sets of genes. A third and more recently discovered mode of regulation is illustrated by non-coding regulatory RNAs. This mode of regulation has been found in all kingdoms of life. In E. coli, about a hundred of these RNA have been identified so far. They are usually referred to as small RNAs (sRNAs) because of their short size (typically around 100 nts) and a large group is involved in post-transcriptional control of the expression of target-genes. Interestingly, several laboratories have shown that the synthesis of E. coli outer membrane proteins, the first to "sense" the environment, are often subjected to sRNA regulation.
In the recent years, it has become clear that these three modes of regulation are not independent. We have ourselves found that the EnvZ-OmpR TCS, known to respond to osmotic changes, activates OmrA and OmrB (OmrA/B), two homologous sRNAs that repress the expression of several genes encoding membrane proteins. Interestingly, these two sRNAs also repress the synthesis of EnvZ-OmpR, creating a regulatory feedback loop. More recently, we found that the MicA sRNA, originally identified as a regulator of OmpA, an abundant porin, negatively regulates the expression of the PhoPQ TCS. This not yet published result is interesting for several reasons. First because the expression of MicA is regulated by the alternative sigma factor ?E, known to respond to envelope stresses. Our finding thus links three regulatory modes, sRNAs, sigma factors and TCS-dependent regulation. In addition, PhoPQ is an important TCS, extremely well studied in S. typhimurium, that is involved in very different biological phenomena such as Mg2+ homeostasis and virulence.
The project proposed here focuses on the study of OmrA/B, MicA and their TCS targets. More precisely, as our preliminary data strongly suggest that additional sRNAs are involved in regulation of phoPQ expression, we will aim at identifying these sRNAs by genetic approaches. Conversely, we will also try to identify other targets of OmrA/B using transcriptomics, proteomics and also by developing an in vivo system to detect and analyze RNA-RNA interactions. If successful, this system could have multiple applications. We will also address the question of how these sRNAs regulate the expression of their targets. In particular, since PhoPQ TCS is involved in Mg2+ homeostasis, we will investigate whether regulation of phoPQ by MicA (and possibly other sRNAs) shows any particular dependence to this essential divalent cation. Finally, we will focus on the connections between the OmrA/B- and MicA-dependent loops and more generally on the relationship between TCS, sRNAs and sigma factors.
The coordinator of this project, Maude Guillier, was a post-doctoral fellow in the laboratory of Susan Gottesman. During her post-doc, she worked mainly on OmrA/B related phenomena. She joined the UPR9073 of the CNRS at the IBPC in September 2008, where she is now heading a small group who discovered the involvement of sRNAs in phoPQ regulation. The project proposed here is based on this discovery and on preliminary results obtained during her post-doc and would be performed entirely by her group.