The <i>Bacillus subtilis</i> endospore crust: protein interaction network, architecture and glycosylation state of a potential glycoprotein layer

Bartels, Julia; Blüher, Anja; Castellanos, Sebastián López; Richter, Marcus; Günther, Markus; Mascher, Thorsten

doi:10.1111/mmi.14381

Cited by 24 publications

(48 citation statements)

References 42 publications

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“…These polysaccharides, without the modifications generated by SwsB, would impair CwlJ activity. Indeed, bioinformatic analyses have found several putative glycosyltransferases that are predicted to be under SigK and SigE control (53). Some of these are responsible for glycosylating proteins in the spore crust, while others are of unknown function.…”

Section: Discussionmentioning

confidence: 99%

SwsB and SafA Are Required for CwlJ-Dependent Spore Germination in Bacillus subtilis

et al. 2020

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When Bacillus subtilis spores detect nutrients, they exit dormancy through the processes of germination and outgrowth. A key step in germination is the activation of two functionally redundant cell wall hydrolases (SleB and CwlJ) that degrade the specialized cortex peptidoglycan that surrounds the spore. How these enzymes are regulated remains poorly understood. To identify additional factors that affect their activity, we used transposon sequencing to screen for synthetic germination defects in spores lacking SleB or CwlJ. Other than the previously characterized protein YpeB, no additional factors were found to be specifically required for SleB activity. In contrast, our screen identified SafA and YlxY (renamed SwsB) in addition to the known factors GerQ and CotE as proteins required for CwlJ function. SafA is a member of the spore’s proteinaceous coat and we show that, like GerQ and CotE, it is required for accumulation and retention of CwlJ in the dormant spore. SwsB is broadly conserved among spore formers, and we show that it is required for CwlJ to efficiently degrade the cortex during germination. Intriguingly, SwsB resembles polysaccharide deacetylases, and its putative catalytic residues are required for its role in germination. However, we find no chemical signature of its activity on the spore cortex or in vitro. While the precise, mechanistic role of SwsB remains unknown, we explore and discuss potential activities. IMPORTANCE Spore formation in Bacillus subtilis has been studied for over half a century, and virtually every step in this developmental process has been characterized in molecular detail. In contrast, how spores exit dormancy remains less well understood. A key step in germination is the degradation of the specialized cell wall surrounding the spore called the cortex. Two enzymes (SleB and CwlJ) specifically target this protective layer, but how they are regulated and whether additional factors promote their activity are unknown. Here, we identified the coat protein SafA and a conserved but uncharacterized protein YlxY as additional factors required for CwlJ-dependent degradation of the cortex. Our analysis provides a more complete picture of this essential step in the exit from dormancy.

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Section: Discussionmentioning

confidence: 99%

SwsB and SafA Are Required for CwlJ-Dependent Spore Germination in Bacillus subtilis

et al. 2020

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“…The crust structure is mainly provided by the CotV, CotX, and CotY proteins, among which, CotY would be the major structural component (15). Interestingly, CotV and CotX share homologue domains and putative N-glycosylation motifs, which make these proteins candidates for glycosylation (15). McKenney and colleagues suggested that CotX, CotY, and CotZ are the crust morphogenetic proteins (12), and recent studies have confirmed that these proteins play a major role in crust assembly (14,15).…”

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confidence: 99%

“…Interestingly, CotV and CotX share homologue domains and putative N-glycosylation motifs, which make these proteins candidates for glycosylation (15). McKenney and colleagues suggested that CotX, CotY, and CotZ are the crust morphogenetic proteins (12), and recent studies have confirmed that these proteins play a major role in crust assembly (14,15). CotZ could be the main morphogenetic protein, since it is involved in the proper localization of most of the crust proteins (14).…”

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confidence: 99%

“…CotZ could be the main morphogenetic protein, since it is involved in the proper localization of most of the crust proteins (14). CotV could also be involved in the crust assembly by propagating the crust structure from the polar cap-like structure to the middle part of the spore (15). CotZ, which required CotE and CotO but none of the crust proteins for localization, seems to have an important anchoring function for the crust in addition to its assembly function (15).…”

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confidence: 99%

“…CotV could also be involved in the crust assembly by propagating the crust structure from the polar cap-like structure to the middle part of the spore (15). CotZ, which required CotE and CotO but none of the crust proteins for localization, seems to have an important anchoring function for the crust in addition to its assembly function (15). CotW plays a role in maintaining the structural integrity of the crust and might play an anchoring role at the interface of crust and coat (14,15).…”

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confidence: 99%

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The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

Dubois

Krzewinski

Yamakawa

et al. 2020

mBio

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The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni. We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis. Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge. IMPORTANCE Bacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

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Impact of bacterial admixtures on the compressive and tensile strengths, permeability, and pore structure of ternary mortars: Comparative study of ureolytic and phototrophic bacteria

Aceituno‐Caicedo,

Shvarzman,

Zhutovsky

et al. 2023

Biotechnology Journal

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The addition of bacterial biomass to cementitious materials can improve strength and permeability properties by altering the pore structure. Photoautotrophic bacteria are understudied mortar bio‐additives that do not produce unwanted by‐products compared to commonly studied ureolytic species. This study directly compares the impact of the addition of heterotrophic Bacillus subtilis to photoautotrophic Synechocystis sp. PCC6803 on mortar properties and microstructure. Cellulose fibers were used as a bacteria carrier. A commercial concrete healing agent composed of dormant bacterial spores was also tested. Strength, water absorption tests, mercury intrusion porosimetry, differential scanning calorimetry, thermogravimetric analysis, and scanning electron microscopy were applied to experimental mortar properties. The photoautotrophic modifications had a stronger positive impact on mortar strength and permeability properties than sporulated heterotrophic modifications due to differences in surface properties and production of exopolysaccharides. The findings provide support for photoautotrophic species as additives for mortars to move away from ammonia‐generating species.

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The Bacillus subtilis endospore crust: protein interaction network, architecture and glycosylation state of a potential glycoprotein layer

Cited by 24 publications

References 42 publications

SwsB and SafA Are Required for CwlJ-Dependent Spore Germination in Bacillus subtilis

SwsB and SafA Are Required for CwlJ-Dependent Spore Germination in Bacillus subtilis

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

Impact of bacterial admixtures on the compressive and tensile strengths, permeability, and pore structure of ternary mortars: Comparative study of ureolytic and phototrophic bacteria

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