Kit J. Pogliano
Phagocytosis and Sporulation in Bascillus subtilis
Bacillus subtilis is a gram positive bacterium with a simple developmental pathway leading to spore formation under conditions of nutrient limitation. Shortly after the formation of the two cells required for spore formation, the septum between them begins to migrate around the forespore, ultimately pinching it off as a free protoplast within the mother-cell cytoplasm (see Figure 1). This process, known as engulfment, demonstrates that B. subtilis is capable of moving macromolecules (in this case the cell envelope) from one region of the cell to another. Such abilities are not normally considered part of the bacterial repertoire, but it has recently been recognized that bacterial chromosomes are actively pulled apart prior to division, suggesting the existence of a mitotic apparatus in bacteria. The nature of this apparatus, like that used to drive the phagocytosis-like process of engulfment, is entirely unclear.
My lab seeks to understand the mechanism of engulfment, as a model system for understanding how bacteria move macromolecules within their cells. We have developed methods to allow the observation of engulfment in living bacteria (Figure 1). Our research is focused on three main questions: What is the mechanism of engulfment? A crucial first step towards understanding the mechanism of engulfment is the identification of the proteins required for engulfment, a careful dissection of the effects of their absence, and the identification of proteins with which they interact. We are therefore searching for additional engulfment proteins using both genetic and biochemical approaches.
How do bacteria catalyze membrane fusion? The final step of engulfment is the fusion of the engulfing membranes at the distal side of the forespore. We have developed an in vivo assay for membrane fusion during engulfment, and have isolated mutants that inhibit membrane fusion. We predict that in bacterial cells, as in eukaryotic cells, membrane fusion will be both tightly regulated and catalyzed.
How are bacterial cells organized? Subcellular protein targeting is crucial for many essential processes in bacteria, and is also crucial for engulfment. We are investigating the subcellular localization of engulfment and membrane fusion proteins.
Figure 1, Foreground: Time-lapse deconvolution microscopy showing the process of engulfment in a single sporangium whose membranes have been stained with FM4-64. At the onset of engulfment (left image), two adjacent cells are present, the smaller forespore and the larger mother cell. During engulfment, the larger mother cell engulfs the forespore (middle images). After the completion of engulfment (right image), the forespore lies within the cytoplasm of the mother cell, where it will develop into a dormant spore.
Background: A sporulating culture of B. subtilis stained with FM4-64 (red) to reveal the membranes. Various stages of engulfment can been seen in the culture as well as vegetative cells containing medial septa.