EXStasis

Organisms across all kingdoms of life have evolved the ability to turn on specific genetic differentiation programs that confer new properties, such as those defining the different stages of development of multicellular eukaryotic organisms. A well-known and widespread prokaryotic differentiation program is competence for genetic transformation, which provides the cells with the ability to capture, internalize and incorporate exogenous DNA into their genome at sites of homology. As such, this horizontal gene transfer process promotes acquisition of new genetic traits and, consequently, drives evolution of these single-celled microorganisms. The DNA-uptake and processing mechanisms of transformation are conserved among bacteria and rely on some few common effectors that work coordinately with several other species-specific components. Competence is a regulated property and the molecular rules governing its development appear to vary considerably among species. Altogether, competence for genetic transformation appears to be tightly integrated into each particular bacterial life-style and represents a major aspect of their physiology.

How competence for genetic transformation is coordinated with other events of the cell cycle remains largely unknown. Here, we will address this question in the major human pathogen Streptococcus pneumoniae (the Pneumococcus), one of the best known and most flexible models for the study of competence for transformation. Remarkably, pneumococcal competence, also termed the X-state, develops abruptly and simultaneously in all cells of an exponentially growing culture and is maintained for a short period of about 20 minutes before rapidly decaying. During this transient period, the cell synthesizes most pieces of the transformasome, a large multiprotein machinery that directs transformation via an ordered series of reactions driving the binding, internalization and integration of exogenous DNA into the recipient genome by homologous recombination. We have previously shown that transformasome assembly in growing pneumococcal cells take place in the cytoplasmic membrane at midcell, the division site. Remarkably, cell growth is transiently delayed in competent pneumococcal cultures. Using single-cell imaging analysis, we found that the cell constriction process is also delayed in competent cells. We inferred that this arrest could reflect the establishment of a cell-division checkpoint that couples the transformation process with the cell cycle, or an interference with cell division owing to assembly of the transformasome at the septum. Another explanation would be that competent pneumococci spatially coordinate remodelling of peptidoglycan with assembly of the transformasome to facilitate its protrusion through the cell wall.

The general scientific objectives of this project, named EXStasis for “Exploring the X-state cellular Stasis”, are to discover how competence for genetic transformation provokes a growth arrest in pneumococci and how transformasome assembly is targeted and proceeds at the division site of growing cells. EXStasis is organized into 3 tasks with the goals of understanding the mechanisms developed during pneumococcal competence to take control of the cell-cycle and to assemble a large, membrane-bound machinery spanning the septal membrane and the cell wall at midcell without damaging cell integrity or ability to generate daughter cells. We will address these issues at the molecular level in living cells, by using a multidisciplinary approach combining molecular genetics and proteomics, together with state of the art imaging techniques of different levels of resolution

Ultimately, studying how S. pneumoniae coordinates transformation with its life cycle will provide new clues as to the role(s) of competence and transformation in the biology of this pathogenic bacterium.

Collaborators:

Rut Carballido-López: ProCeD - MICALIS – INRA – Jouy-en-Josas

Christophe Grangeasse: MMSB - Molecular Microbiology and Structural Biochemistry – CNRS - Lyon

Funding