Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Endospore formation by<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Bacillus subtilis</jats:named-content>is a complex and dynamic process. One of the major challenges of sporulation is the assembly of a protective, multilayered, proteinaceous spore coat, composed of at least 70 different proteins. Spore coat formation can be divided into two distinct stages. The first is the recruitment of proteins to the spore surface, dependent on the morphogenetic protein SpoIVA. The second step, known as encasement, involves the migration of the coat proteins around the circumference of the spore in successive waves, a process dependent on the morphogenetic protein SpoVID and the transcriptional regulation of individual coat genes. We provide genetic and biochemical evidence supporting the hypothesis that SpoVID promotes encasement of the spore by establishing direct protein-protein interactions with other coat morphogenetic proteins. It was previously demonstrated that SpoVID directly interacts with SpoIVA and the inner coat morphogenetic protein, SafA. Here, we show by yeast two-hybrid and pulldown assays that SpoVID also interacts directly with the outer coat morphogenetic protein, CotE. Furthermore, by mutational analysis, we identified a specific residue in the N-terminal domain of SpoVID that is essential for the interaction with CotE but dispensable for the interaction with SafA. We propose an updated model of coat assembly and spore encasement that incorporates several physical interactions between the principal coat morphogenetic proteins.</jats:p>

SpoIVA and SipL Are Clostridium difficile Spore Morphogenetic Proteins

“…mutants (18,19,39). This observation suggests that SpoIVA does not link coat and cortex formation in C. difficile as it does in B. subtilis (18,25,40).…”

“…While gene-specific primers for sipL were sufficient to amplify across the targetron sequence, targetron-specific primers CDEP1138 and CDEP692, which bind to the intron insertion, were used in conjunction with spoIVA-specific primers in two separate PCRs to visualize the targeted insertion into spoIVA. (23,25) and the SpoIVA binding domain (red rectangles) (23,24). Only the C-terminal LysM domain (black rectangles) of SpoVID exhibits significant homology to SipL.…”

“…Although SpoIVA initially localizes to a cap at the mother-cell-proximal pole of the forespore (16), SpoIVA can fully encase the forespore only in the presence of SpoVID (23). SpoVID also recruits additional proteins to the growing basement layer and thus plays a critical role in coat assembly in B. subtilis (16,23,(25)(26)(27).…”

Physical interaction and assembly of Bacillus subtilis spore coat proteins CotE and CotZ studied by atomic force microscopy

“…SpoIVA which assembles in the coat basement layer with the assistance of SpoVM, a small amphipathic a-helical peptide, plays a key role in localization of other coat protein components on the spore surface (Ramamurthi et al, 2006). SpoVID along with SpoVM are necessary for the spore encasement during which the spore becomes fully surrounded by coat proteins (de Francesco et al, 2012;Wang et al, 2009). SafA is the major inner coat morphogenetic protein and contributes to patterning of the coat structure (McKenney et al, 2010;Ozin et al, 2000Ozin et al, , 2001.…”

CotG-Like Modular Proteins Are Common among Spore-Forming Bacilli

“…At least some coat proteins are subject to posttranslational maturation events, including proteolytic cleavage, cross-linking, phosphorylation, and glycosylation reactions (10). An important subset of coat proteins, referred to as morphogenetic proteins, controls the assembly of other coat components and the formation of coat and crust layers (11)(12)(13)(14)(15). The subset of morphogenetic coat proteins includes major members such as SpoIVA, SafA, SpoVID, and CotE, which are needed to drive coat and crust formation, and minor members responsible for the maturation or assembly of some other coat proteins (6,10).…”