Dataset Information

Searching for protein-protein interactions within the Bacillus subtilis spore coat.

ABSTRACT: The capability of endospores of Bacillus subtilis to withstand extreme environmental conditions is secured by several attributes. One of them, the protein shell that encases the spore and is known as the coat, provides the spore with its characteristic resistance to toxic chemicals, lytic enzymes, and predation by unicellular and multicellular eukaryotes. Despite most of the components of the spore coat having been identified, we have only a vague understanding of how such a complex structure is assembled. Using the yeast two-hybrid system, we attempted to identify direct contacts among the proteins allocated to the insoluble fraction of the spore coat: CotV, CotW, CotX, CotY, and CotZ. We also examined whether they could interact with CotE, one of the most crucial morphogenetic proteins governing outer coat formation and also present in the insoluble fraction. Out of all 21 possible interactions we tested, 4 were found to be positive. Among these interactions, we confirmed the previous observation that CotE forms homo-oligomers. In addition, we observed homotypic interactions of CotY, strong interactions between CotZ and CotY, and relatively weak, yet significant, interactions between CotV and CotW. The results of this yeast two-hybrid analysis were confirmed by size exclusion chromatography of recombinant coat proteins and a pull-down assay.

SUBMITTER: Krajcikova D

PROVIDER: S-EPMC2687167 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Searching for protein-protein interactions within the Bacillus subtilis spore coat.

Krajcíková Daniela D Lukácová Magda M Müllerová Denisa D Cutting Simon M SM Barák Imrich I

Journal of bacteriology 20090320 10

The capability of endospores of Bacillus subtilis to withstand extreme environmental conditions is secured by several attributes. One of them, the protein shell that encases the spore and is known as the coat, provides the spore with its characteristic resistance to toxic chemicals, lytic enzymes, and predation by unicellular and multicellular eukaryotes. Despite most of the components of the spore coat having been identified, we have only a vague understanding of how such a complex structure is ...[more]

PMID: 19304857

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Project description:At a late stage in spore development in Bacillus subtilis, the mother cell directs synthesis of a layer of peptidoglycan known as the cortex between the two forespore membranes, as well as the assembly of a protective protein coat at the surface of the forespore outer membrane. SafA, the key determinant of inner coat assembly, is first recruited to the surface of the developing spore and then encases the spore under the control of the morphogenetic protein SpoVID. SafA has a LysM peptidoglycan-binding domain, SafALysM, and localizes to the cortex-coat interface in mature spores. SafALysM is followed by a region, A, required for an interaction with SpoVID and encasement. We now show that residues D10 and N30 in SafALysM, while involved in the interaction with peptidoglycan, are also required for the interaction with SpoVID and encasement. We further show that single alanine substitutions on residues S11, L12, and I39 of SafALysM that strongly impair binding to purified cortex peptidoglycan affect a later stage in the localization of SafA that is also dependent on the activity of SpoVE, a transglycosylase required for cortex formation. The assembly of SafA thus involves sequential protein-protein and protein-peptidoglycan interactions, mediated by the LysM domain, which are required first for encasement then for the final localization of the protein in mature spores.IMPORTANCE Bacillus subtilis spores are encased in a multiprotein coat that surrounds an underlying peptidoglycan layer, the cortex. How the connection between the two layers is enforced is not well established. Here, we elucidate the role of the peptidoglycan-binding LysM domain, present in two proteins, SafA and SpoVID, that govern the localization of additional proteins to the coat. We found that SafALysM is a protein-protein interaction module during the early stages of coat assembly and a cortex-binding module at late stages in morphogenesis, with the cortex-binding function promoting a tight connection between the cortex and the coat. In contrast, SpoVIDLysM functions only as a protein-protein interaction domain that targets SpoVID to the spore surface at the onset of coat assembly.

Dataset Information

Searching for protein-protein interactions within the Bacillus subtilis spore coat.

Publications

Searching for protein-protein interactions within the Bacillus subtilis spore coat.

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