Talks by Alexandre D’HALLUIN and Grégory BOËL

Alexandre D’HALLUIN
Ciarán Condon’s lab, EGM CNRS, Université de Paris-Cité, Institut de Biologie Physico-Chimique

Title:
Embedding a ribonuclease in the spore crust couples gene expression to spore development in Bacillus subtilis.

Abstract:
Sporulation is an irreversible pathway conserved across firmicutes which leads to the formation of a highly resistant and dormant bacterial state called endospore, allowing the bacteria to survive to extreme environmental conditions for millions of years (1-4). The agent of anthrax, botulism or tetanus have integrated spore formation in their pathogenic cycle, making it a critical cell development to study in medical research. In the model organism B. subtilis, sporulation allows the bacteria to resist to high stress conditions such as nutrient deprivation, heat, desiccation or radiation. While sporulation is well studied, it has been shown some RNases are important for proper sporulation, while their mechanism and function during sporulation are poorly understood (5-7). Bacterial RNA degradation is both important for the recycling ribonucleotides and controlling gene expression through a variety of cleavage mechanisms (8,9). The activity of some ribonucleases and the accessibility of their RNA substrates are regulated under specific stress conditions, such as nutrient starvation or amino-acid depletion, to allow a tight control of RNA decay and maturation (8, 10). Here we present KapD, a ribonuclease exhibiting a 3’-exonuclease activity and specifically expressed during sporulation of B. subtilis. KapD is regulated by the sporulation specific sigma factors E and K, allowing expression within the mother cell at the early and late stage of sporulation (2). Deletion of the kapD locus or inactivation of its catalytic site strongly decreases the adhesiveness of the outer coat and crust layers of the endospore, suggesting that its ribonuclease activity is essential for the correct formation of the spore morphological structure and resistance. Using dual-color fluorescent microscopy, we show a dynamic assembly of the protein into the spore crust layer in a similar way to outer spore coat proteins. With yeast two-hybrid assay and in vitro validations, we showed that KapD interacts with the well-known crust protein CotY, accounting for its localization in the outermost spore layer. This interaction inhibits the catalytic activity of KapD. Coupling proteomic analysis with Northern Blot, we identified a key developmental transcriptional regulator sigK mRNA as the main substrate of KapD. In fine, we provide evidence that KapD controls the timing of expression and activation of SigK prior to its sequestration within the spore crust layer, adding another level of regulation of SigK expression.

Grégory BOËL
UMR8261 CNRS / Université Paris Cité Institut de Biologie Physico-Chimique

Title:
Completing the picture of the translation apparatus: ABC-F proteins at the rescue of the ribosome.

Abstract:
The canonical bacterial translation apparatus, established since the 1970s, has enabled the development of minimal systems for in vitro protein production. However, this core machinery remains incomplete, requiring additional translation factors to assist ribosomes in synthesizing specific amino acid sequences. Our research focuses on a novel family of translation factors (TFs) known as the ABC-F protein family which is conserved from bacteria to humans. We have discovered that these TFs can rescue ribosomes stalled during the polymerization of specific sequences or in response to antibiotics. Specifically, we elucidate the unique physiological function of one member, EttA, in facilitating the ribosomal synthesis of proteins with poly-acidic residues early in the sequence. Furthermore, we demonstrate its impact on the global regulation of gene expression in Escherichia coli. This newly characterized function unifies the roles of ABC-F factors and uncovers a new level of gene expression regulation in bacteria.