Expression of
            <i>yeeK</i>
            during
            <i>Bacillus subtilis</i>
            Sporulation and Localization of YeeK to the Inner Spore Coat using Fluorescence Microscopy

Takamatsu, Hiromu; Imamura, Daisuke; Kuwana, Ritsuko; Watabe, Kazuhito

doi:10.1128/jb.01269-08

Cited by 15 publications

(10 citation statements)

References 54 publications

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“…The strong dependency on SafA indicates that the main location of Tgl in the coat is its inner substructure. This is consistent with the localization of SafA FL , C30, and the two other known Tgl substrates, GerQ and YeeK, with the increased extractability of Tgl following lysozyme treatment, and finally with the immunogold labeling data (39,49,63, and this work).…”

Section: Discussionsupporting

confidence: 90%

Autoregulation of SafA Assembly through Recruitment of a Protein Cross-Linking Enzyme

2018

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The coat of spores is a multiprotein protective structure that also arbitrates many of the environmental interactions of the spore. The coat assembles around the cortex peptidoglycan layer and is differentiated into an inner and an outer layer and a crust. SafA governs assembly of the inner coat, whereas CotE drives outer coat assembly. SafA localizes to the cortex-coat interface. Both SafA and its short form C30 are substrates for Tgl, a coat-associated transglutaminase that cross-links proteins through ε-(γ-glutamyl)lysyl isopeptide bonds. We show that SafA and C30 are distributed between the coat and cortex layers. The deletion of increases the extractability of SafA, mainly from the cortex. Tgl itself is mostly located in the inner coat and cortex. The localization of Tgl-cyan fluorescent protein (Tgl-CFP) is strongly, but not exclusively, dependent on However, the association of Tgl with the cortex requires Together, our results suggest an assembly pathway in which Tgl is first recruited to the forming spore in a manner that is only partially dependent on SafA and then is drafted to the cortex by SafA. Tgl, in turn, promotes the conversion of coat- and cortex-associated SafA into forms that resist extraction, possibly by catalyzing the cross-linking of SafA to other coat proteins, to the cortex, and/or to cortex-associated proteins. Therefore, the final assembly state of SafA relies on an autoregulatory pathway that requires the subcellular localization of a protein cross-linking enzyme. Tgl most likely exerts a "spotwelding" activity, cross-linking preformed complexes in the cortex and inner coat layers of spores. In this work, we show how two proteins work together to determine their subcellular location within the coat of bacterial endospores. endospores are surrounded by a multilayer protein coat composed of over 80 proteins, which surrounds an underlying peptidoglycan layer (the spore cortex) protecting it from lytic enzymes. How specific coat proteins are targeted to specific layers of the coat is not well understood. We found that the protein SafA recruits a protein-cross-linking enzyme (a transglutaminase) to the cortex and inner layers of the coat, where both are cemented, by cross-linking, into macromolecular complexes.

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Section: Discussionsupporting

confidence: 90%

Autoregulation of SafA Assembly through Recruitment of a Protein Cross-Linking Enzyme

2018

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“…YeeK fusions produced a smaller diameter than the other proteins. These results are consistent with previous reports that CotA and CotB are located on the outer surfaces of spores and that YeeK is located in the inner coat (15,44,45). Since the diameters measured with GFPfused proteins were typically slightly longer (up to 1 pixel) than those measured with the corresponding RFP-fused proteins, small differences in relative localizations are not very reliable.…”

Section: Resultssupporting

confidence: 80%

“…S1 in the supplemental material). The assembly of YmaG, YsnD, and YeeK was shown previously to be independent of CotE (17,44). The CotE independence of CotD assembly was demonstrated previously by SDS-PAGE (52).…”

Section: Resultsmentioning

confidence: 79%

“…However, cotE disruption disrupts the outer coat layer and also affects the assembly of some inner coat proteins (17), so assignments based on CotE dependence are not definitive. Many recent studies of protein localization have exploited fluorescence microscopy with fluorescent proteins, but these methods have not been used to distinguish inner and outer spore coat proteins until recently because the resolution was insufficient (44). In this study, measurements of the diameters of the fluorescent spore coat protein layers of many spores achieved sufficient resolution to distinguish inner and outer spore coats.…”

Section: Discussionmentioning

confidence: 83%

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Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats

et al. 2010

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Bacterial spores are encased in a multilayered proteinaceous shell known as the coat. In Bacillus subtilis, over 50 proteins are involved in spore coat assembly but the locations of these proteins in the spore coat are poorly understood. Here, we describe methods to estimate the positions of protein fusions to fluorescent proteins in the spore coat by using fluorescence microscopy. Our investigation suggested that CotD, CotF, CotT, GerQ, YaaH, YeeK, YmaG, YsnD, and YxeE are present in the inner coat and that CotA, CotB, CotC, and YtxO reside in the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat and were more abundant at the mother cell proximal pole of the forespore, whereas CotA and CotC were more abundant at the mother cell distal pole of the forespore. These polar localizations were observed both in sporangia prior to the release of the forespore from the mother cell and in mature spores after release. Moreover, CotB was observed at the middle of the spore as a ring-or spiral-like structure. Formation of this structure required cotG expression. Thus, we conclude not only that the spore coat is a multilayered assembly but also that it exhibits uneven spatial distribution of particular proteins.Proper localization and assembly of proteins in cells and subcellular structures are essential features of living organisms. Complex protein assemblies, including ribosomes, flagella, and the cytokinetic machinery, play important roles in bacteria (26,27,40). Studying how these complex structures are formed is a fundamental theme in molecular biology. In this work, we developed a method to analyze one of the most complex bacterial protein assemblies: the spore coat of Bacillus subtilis.Sporulation of B. subtilis is initiated in response to nutrient limitation, and it involves a highly ordered program of gene expression and morphological change (33, 42). The first morphological change of sporulation is the appearance of an asymmetrically positioned septum that divides the cell into a larger mother cell and a smaller forespore. Next, the mother cell membrane migrates around the forespore membrane during a phagocytosislike process called engulfment. The completion of engulfment involves fusion of the mother cell membrane to pinch off the forespore within the mother cell. Compartment-specific gene expression brings about maturation of the spore and its release upon lysis of the mother cell (reviewed in reference 19). Mature spores remain viable during long periods of starvation and are resistant to heat, toxic chemicals, lytic enzymes, and other factors capable of damaging vegetative cells (30). Spores germinate and resume growth when nutrients become available (32).The outer portions of Bacillus spores consist of a cortex, a spore coat layer, and in some cases, an exosporium. The cortex, a thick layer of peptidoglycan, is deposited between the inner and the outer membranes of the forespore, and it is responsible for maintaining the highly dehydrated state of the core, thereby contri...

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“…Coat proteins are generally not conserved between spore‐forming species, and their identification has traditionally been technically cumbersome. However, the use of fluorescent microscopy in combination with genomic approaches (Kim et al ., ; Takamatsu et al ., ) has greatly facilitated the identification of additional coat proteins. A recent extension of this work is the application of sophisticated image analysis to the localization of fluorescent fusions that allowed direct assignment of proteins to a particular coat layer, as well as the identification of a previously undescribed layer (McKenney et al ., ).…”

Section: Spore Morphogenesismentioning

confidence: 99%

Recent progress inBacillus subtilissporulation

2012

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The Gram-positive bacterium Bacillus subtilis can initiate the process of sporulation under conditions of nutrient limitation. Here, we review some of the last five years of work in this area, with a particular focus on the decision to initiate sporulation, DNA translocation, cell-cell communication, protein localization and spore morphogenesis. The progress we describe has implications not just for the study of sporulation but also for other biological systems where homologs of sporulation-specific proteins are involved in vegetative growth.

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Expression of yeeK during Bacillus subtilis Sporulation and Localization of YeeK to the Inner Spore Coat using Fluorescence Microscopy

Cited by 15 publications

References 54 publications

Autoregulation of SafA Assembly through Recruitment of a Protein Cross-Linking Enzyme

Autoregulation of SafA Assembly through Recruitment of a Protein Cross-Linking Enzyme

Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats

Recent progress inBacillus subtilissporulation

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