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Research in the Rudner Lab:
An introduction to spore formation in B. subtilis

Figure 1
Figure 1.

In response to nutrient deprivation (starvation) the rod-shaped soil bacterium Bacillus subtilis differentiates into a dormant, stress-resistant spore (Fig. 1). B. subtilis spores can withstand extreme heat, radiation, chemical assaults and time (spores can remain dormant for more than a million years).

A 1ml culture of wild-type B. subtilis produces ~5x108 spores. These spores can easily withstand 80°C for 20 minutes while vegetative (non-sporulated) cells are killed at this temperature. Thus, the study of sporulation is grounded in an assay that is quantitative over 8 logs.
Figure 2
Figure 2.

The major morphological events in the process of sporulation are outlined in Figure 2. Upon commitment to sporulate an asymmetric division partitions the developing cell (the sporangium) into a large cell (the mother cell) and a small cell (the prospective spore or forespore). Initially, the two cells lie side-by-side but shortly after polar division the mother cell membranes migrate around the forespore generating a cell-within-a-cell. As a result of this engulfment process the forespore is surrounded by two membranes: its own (called the inner forespore membrane) and a second membrane derived from the mother cell (called the outer forespore membrane). At this stage in development the mother cell nurtures the forespore, packaging it in a protective protein coat while the forespore prepares for dormancy. Once the spore is fully mature, it is released into the environment through lysis of the mother cell.

Figure 3
Figure 3.

During this developmental process a cascade of alternative transcription factors are activated in a stage- and compartment-specific manner (Fig. 3). Shortly after polar division the first compartment-specific transcription factor (σF) is activated in the forespore. Soon afterwards σE is activated in the mother cell. Upon completion of engulfment the second forespore-specific transcription factor σG is activated. Shortly after σK is activated in the mother cell.

Figure 4
Figure 4.

We can easily visualize this compartment-specific gene expression by fusing different promoters to the gfp (Fig. 4). Thus, the mother cell and forespore follow completely different programs of developmental gene expression.
Figure 5
Figure 5.

These two programs are linked to each other by intercellular signal transduction pathways (Fig. 5) to ensure that gene expression in one cell is kept in register with gene expression in the other.

One of the major focuses in our lab is to elucidate the mechanism by which information is transduced across a lipid bilayer. We use the last signal transduction pathway (the activation σK in the mother cell under the control of the σG in the forespore) as our guide.

Figure 6
Figure 6.

Three of the proteins in this signaling pathway localize with exquisite specificity to the mother cell membrane that surrounds the forespore (Fig. 6). A second area of interest in our lab is to define the cellular landmarks that anchor proteins at particular positions in the cell.

Figure 7
Figure 7.

During the process of sporulation the replicated chromosomes are segregated in an unusual manner. First, the replicated chromosomes adopt an elongated structure that extends from pole-to-pole (Fig. 7). This serpentine structure is called the axial filament. As a result of axial filament formation the asymmetric division plane traps a third of the chromosome in the forespore compartment.

Figure 8
Figure 8.

A DNA translocase (SpoIIIE) pumps the remaining two thirds of the chromosome into the forespore (Fig. 8). Thus, in this unusual cell cycle cytokinesis precedes DNA segregation. A third project in the lab is the analysis of the proteins that remodel and segregate the chromosomes during sporulation.