SpoVID Guides SafA to the Spore Coat in Bacillus subtilis

Abstract

Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encases Bacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.Proteins are targeted to specific subcellular locations during the assembly of a variety of bacterial structures. The structures assembled by prokaryotes include, for example, the cell division septum (3), surface protein layers (S-layers) (36), and a number of surface appendages such as flagella (24) and pili (17). Here we are concerned with the assembly of the Bacillus subtilis spore coat, a proteinaceous structure that encases spores (reviewed in references 7 and 16). The B. subtilis spore is a metabolically dormant cell type that is formed as an adaptive response to nutrient depletion (9, 30, 37). The spore coat provides protection against physical and chemical insults and can sense and respond to nutrients that trigger germination (9, 26).Spore morphogenesis commences by the placement of an asymmetric septum that divides the rod-shaped cell into a larger mother cell and a smaller prespore, each containing a copy of the chromosome. The septal membranes migrate around the prespore, in a process known as engulfment, which eventually results in the formation of a free-floating protoplast (the forespore) separated from the mother cell cytoplasm by a double-membrane system. Cell wall material (the cortex) is deposited between the membranes of the forespore (9, 30, 37). A thick coat composed of more than two dozen proteins is assembled around the outer forespore membrane. The majority of the proteins that make up the coat are synthesized in the mother cell under the direction of mother cell-specific RNA polymerase sigma factors (7,16,30).

Assembly of Multiple CotC Forms into the Bacillus subtilis Spore Coat

“…Yeast two-hybrid system. The Matchmaker two-hybrid system (Clontech) was used as described by Ozin et al (23), with only minor modifications. The cotC coding region was amplified by PCR using primer pair C/5/Nco and C2 ( :GAL1 UAS -GAL1 TATA -HIS3) (Clontech) were independently transformed with the pAS2-1 or pACT-2 vector and/or each of the cotC constructs according to the protocols suggested by the manufacturer.…”

“…Additional proteins with morphogenetic functions are needed for coat formation. SpoVID and SafA are made under sigma E control; SpoVID interacts with SafA and directs it to the forming spore and is also required to maintain the CotE ring around the forespore (5,11,23). In contrast CotH is a morphogenetic protein produced under sigma K control that plays a role in outer coat assembly and the lysozyme resistance of the spore and that, in conjunction with CotE, is also responsible for efficient spore germination (21,33).…”

Functional relationship between SpoVIF and GerE in gene regulation during sporulation of Bacillus subtilis

“…5C). In contrast, other proteins that are essential for coat development, such as SpoIVA, SpoVID, YrbA (SafA) and CotE, are localized on the outer surface of the forespore membrane (Price & Losick, 1999;Ozin et al, 2001;Webb et al, 1995). These distinctive localization patterns suggest that SpoVIF is related to the function of GerE in the mother cell compartment.…”

GerE-independent expression of cotH leads to CotC accumulation in the mother cell compartment during Bacillus subtilis sporulation

“…In contrast, the outer coat proteins are assembled on the outside of the CotE structure (Henriques & Moran, 2000;Driks, 2002). Additional proteins with morphogenetic functions are needed for coat formation: SpoVID, produced under s E control, has the dual role of directing SafA to the forming spore and maintaining the CotE ring around the forespore (Henriques & Moran, 2000;Ozin et al, 2001;Driks, 2002); CotH, produced under s K control, plays a role in the assembly of various outer coat components and in the development of the lysozyme resistance typical of the mature spore (Naclerio et al, 1996;Zilhao et al, 1999). Additionally, CotH, in conjunction with CotE, is required for efficient spore germination (Naclerio et al, 1996).…”