Bacillus subtilis

The goal of our research is to achieve a detailed understanding of the phagocytosis-like process of engulfment, a crucial step in the sporulation pathway of Bacillus subtilis. Engulfment provides an ideal model system for the study of how bacteria move macromolecules within their cells, how they target proteins to specific locations, and how they catalyze membrane fusion. Engulfment can be readily observed using fluorescent membrane stains and deconvolution microscopy, which allow the direct visualization of the engulfing membranes and of the membrane fusion event that is the final stage of engulfment. We can watch individual bacteria complete engulfment, and rapidly characterize the effects of mutations and antibiotics on engulfment.

Our research addresses the following questions:

What is the mechanism of engulfment?A crucial first step towards understanding the mechanism of engulfment is the identification of proteins required for engulfment, a careful dissection of the effects of their absence, and the identification of proteins with which they interact. We are using genetic approaches to identify additional engulfment defective mutants, and are also using biochemical approaches to identify proteins that interact with known engulfment proteins.

How do bacteria catalyze membrane fusion? Engulfment provides the first clear and readily studied example of membrane fusion in a bacterium; we have recently devised an in vivo assay for membrane fusion during engulfment, and have isolated mutants that inhibit membrane fusion. Ultimately we will purify the membrane fusion proteins and reconstitute this membrane fusion event in vitro,to allow the biochemical dissection of the mechanism of membrane fusion.

How are bacterial cells organized?Subcellular protein targeting is crucial for many essential processes in bacteria, and is almost certainly required for engulfment. We will study the subcellular localization of engulfment proteins, and screen our collection of mutants for those that inhibit localization.

Engulfment provides a unique system for the study of prokaryotic cell biology. Although non-essential, engulfment requires capabilities that are essential, such as subcellular protein targeting, membrane fusion, and the movement of macromolecules within cells.
 

The following figure depicts the engulfment prosess:
 

Engulfment starts with the thinning of peptidoglycan in the middle of the sporulation septum (StageIIii), a step spatially regulated by SpoIIB (A.R. Perez). Proteins listed above the arrows are essential for engulfment under all conditions, while those below the arrows are not essential for engulfment, either because they contribute to the efficiency of engulfment (i.e: SpoIIB), or because they are required only under certain conditions (i.e: SpoIIQ which is required only when sporulation is induced by a nutrient exhaustion - Y.-L. Sun). SpoIID, SpoIIM and SpoIIP are required for septal thinning to proceed to the edges of the septum, this step allows the mother clle membrane to migrate up the side of the forespore (Stage IIiii). Migration of the engulfing mother cell membrane across the cell pole requires SpoIIQ only under certain culture conditions. Ultimately the engulfing membranes meet at the cell pole (Stage IIiv) and fuse to fully enclose the forespore (Stage III). This membrance fusion event requires SpoIIIE (M. Sharp). Engulfment is closely coupled to cell specific transcription: following the completion of engulfment, sG becomes active in the forespore, while sK becomes active in the mother cell.