Analysis of the Effects of a
            <i>gerP</i>
            Mutation on the Germination of Spores of Bacillus subtilis

Butzin, Xuan Y.; Troiano, Anthony J.; Coleman, William H.; Griffiths, Keren K.; Doona, Christopher J.; Feeherry, Florence E.; Wang, Guiwen; Li, Yongqing; Setlow, Peter

doi:10.1128/jb.01276-12

Cited by 36 publications

(42 citation statements)

References 47 publications

(91 reference statements)

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“…In at least three Bacillus species, mutants lacking one or all of the GerP proteins germinate more slowly than intact spores with exposure to nutrients (36)(37)(38). The gerP phenotype is suppressed either by spore coat/OM removal chemically or in appropriate coat assembly mutants or by using nutrient germinant concentrations far above what are saturating for intact spores (36)(37)(38). These observations suggest that the GerP proteins facilitate nutrient germinant access to GRs in the IM.…”

Section: Major Unanswered Questions About Spore Germination By Nutrientsmentioning

confidence: 66%

“…However, the access of at least dodecylamine is slow in intact spores, as spores lacking most coat proteins and their OM germinate much faster with dodecylamine than do intact spores (42). In at least three Bacillus species, mutants lacking one or all of the GerP proteins germinate more slowly than intact spores with exposure to nutrients (36)(37)(38). The gerP phenotype is suppressed either by spore coat/OM removal chemically or in appropriate coat assembly mutants or by using nutrient germinant concentrations far above what are saturating for intact spores (36)(37)(38).…”

Section: Major Unanswered Questions About Spore Germination By Nutrientsmentioning

confidence: 99%

“…Some germination proteins are not in the IM, including the CLE CwlJ, which is at the cortex-coat boundary, perhaps associated with its partner protein GerQ (33)(34)(35); some SleB is also likely in this region of the spore (4,35). In addition, the multiple small GerP proteins that appear to facilitate the access of a variety of low-molecular-weight germinants to the IM (36)(37)(38) are likely in the spore coat (see below).…”

Section: Overview Of Spore Germinationmentioning

confidence: 99%

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Germination of Spores of Bacillus Species: What We Know and Do Not Know

2014

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Spores of Bacillus species can remain in their dormant and resistant states for years, but exposure to agents such as specific nutrients can cause spores' return to life within minutes in the process of germination. This process requires a number of sporespecific proteins, most of which are in or associated with the inner spore membrane (IM). These proteins include the (i) germinant receptors (GRs) that respond to nutrient germinants, (ii) GerD protein, which is essential for GR-dependent germination, (iii) SpoVA proteins that form a channel in spores' IM through which the spore core's huge depot of dipicolinic acid is released during germination, and (iv) cortex-lytic enzymes (CLEs) that degrade the large peptidoglycan cortex layer, allowing the spore core to take up much water and swell, thus completing spore germination. While much has been learned about nutrient germination, major questions remain unanswered, including the following. T he germination of the dormant and highly resistant spores formed by members of the Firmicutes phylum, in particular bacilli and clostridia, has long been of significant research interest for four major reasons, as follows: (i) fascinating regulatory systems allow such spores to remain in their dormant, resistant state for years and yet return to active growth in minutes; (ii) while spores of most Firmicutes do not cause disease, spores of some bacilli and clostridia cause food spoilage and food-borne disease, as well as human diseases like gas gangrene, tetanus, botulism, anthrax, and pseudomembranous colitis; (iii) spores of Bacillus anthracis are a major bioterrorism threat; and (iv) spores of Clostridium difficile are an emerging public health threat (1-3). Invariably, it is the germination of spores of these organisms that leads to disease or food spoilage, and yet, when spores germinate and lose their dormancy, they lose their extreme resistance properties and become relatively easy to kill. Germination is thus both an essential part of disease pathogenesis or food spoilage and a major weak spot in these organisms' life cycle. Consequently, there has long been applied interest in spore germination, with researchers seeking to understand this process better in order to either prevent spore germination or accelerate it and then kill the newly sensitive germinated spores. This review will concentrate on the germination of spores of bacilli, primarily because of the large amount of detailed knowledge of the germination of spores of these species compared to that of spores of clostridia. However, some of the differences and similarities between the germination of spores of these related genera will also be presented. Most discussion will focus on the germination of the model sporeformer, Bacillus subtilis, although the mechanisms of germination of B. subtilis spores appear to be similar for spores of other bacilli. The properties of the various proteins that are specifically involved in the germination process will not be discussed in great detail, since these have recently be...

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Section: Major Unanswered Questions About Spore Germination By Nutrientsmentioning

confidence: 66%

Section: Major Unanswered Questions About Spore Germination By Nutrientsmentioning

confidence: 99%

Section: Overview Of Spore Germinationmentioning

confidence: 99%

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Germination of Spores of Bacillus Species: What We Know and Do Not Know

2014

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“…However, the exact mechanism restricting dodecylamine access to the spore's inner membrane in intact spores is not yet clear. In spores of Bacillus species, the GerP proteins, a group of small proteins likely present in the spore coat, are thought to be crucial to ensure rapid access of some germinants to spores' IMs (58,59). However, proteins analogous to GerP proteins have not been identified in Clostridium spores.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

et al. 2015

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The Gram-positive spore-forming anaerobe Clostridium difficile is a leading cause of nosocomial diarrhea. Spores of C. difficile initiate infection when triggered to germinate by bile salts in the gastrointestinal tract. We analyzed germination kinetics of individual C. difficile spores using Raman spectroscopy and differential interference contrast (DIC) microscopy. Similar to Bacillus spores, individual C. difficile spores germinating with taurocholate plus glycine began slow leakage of a ϳ15% concentration of a chelate of Ca 2؉ and dipicolinic acid (CaDPA) at a heterogeneous time T 1 , rapidly released CaDPA at T lag , completed CaDPA release at T release , and finished peptidoglycan cortex hydrolysis at T lysis . T 1 and T lag values for individual spores were heterogeneous, but ⌬T release periods (T release ؊ T lag ) were relatively constant. In contrast to Bacillus spores, heat treatment did not stimulate spore germination in the two C. difficile strains tested. C. difficile spores did not germinate with taurocholate or glycine alone, and different bile salts differentially promoted spore germination, with taurocholate and taurodeoxycholate being best. Transient exposure of spores to taurocholate plus glycine was sufficient to commit individual spores to germinate. C. difficile spores did not germinate with CaDPA, in contrast to B. subtilis and C. perfringens spores. However, the detergent dodecylamine induced C. difficile spore germination, and rates were increased by spore coat removal although cortex hydrolysis did not follow T release , in contrast with B. subtilis. C. difficile spores lacking the cortex-lytic enzyme, SleC, germinated extremely poorly, and cortex hydrolysis was not observed in the few sleC spores that partially germinated. Overall, these findings indicate that C. difficile and B. subtilis spore germination exhibit key differences. IMPORTANCESpores of the Gram-positive anaerobe Clostridium difficile are responsible for initiating infection by this important nosocomial pathogen. When exposed to germinants such as bile salts, C. difficile spores return to life through germination in the gastrointestinal tract and cause disease, but their germination has been studied only with population-wide measurements. In this work we used Raman spectroscopy and DIC microscopy to monitor the kinetics of germination of individual C. difficile spores, the commitment of spores to germination, and the effect of germinant type and concentration, sublethal heat shock, and spore decoating on germination. Our data suggest that the order of germination events in C. difficile spores differs from that in Bacillus spores and provide new insights into C. difficile spore germination. C lostridium difficile is a Gram-positive, spore-forming, strictly anaerobic bacterium that has become a leading cause of antibiotic-associated diarrhea worldwide (1). Antibiotic treatment disrupts the normal colonic flora that typically suppresses C. difficile growth and allows ingested C. difficile spores to germinate, outgrow...

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“…Indeed, these outer layers appear to be capable of restricting access of even low-molecular-weight nutrient germinants such as L-alanine to the spores' GRs in the IM (36,37). Consequently, we began by measuring levels of biotinylated proteins generated upon incubation of the biotinylation reagent with either chemically decoated wild-type spores (strain PS533) or ⌬cotE ⌬gerE strain spores (strain PS4150), as these types of spores lack both an outer membrane and most of the spore coat protein (10,22), and examining proteins in the IM fraction ( Fig.…”

Section: Procedures For Analysis Of Germination Protein Topologymentioning

confidence: 99%

Topology and Accessibility of Germination Proteins in the Bacillus subtilis Spore Inner Membrane

Korza

Setlow

2013

J Bacteriol

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Access to a membrane-impermeant biotinylation reagent as well as protease sensitivity was used to determine germination proteins' topology in the inner membrane (IM) of decoated dormant spores and intact germinated Bacillus subtilis spores. The proteins examined were four nutrient germinant receptor (GR) subunits, the GerD protein, essential for normal GR-dependent spore germination, the SpoVAD protein, essential for dipicolinic acid movement across the IM, the SleB cortex-lytic enzyme, and the YpeB protein, essential for SleB assembly in spores, as well as green fluorescent protein (GFP) in the spore core. GerD and SpoVAD as well as GFP in the spore were not biotinylated in decoated dormant spores. However, GR subunits, SleB, and YpeB were biotinylated 4 to 36% in decoated dormant spores, although these levels were not increased by higher biotinylation reagent concentrations or longer reaction times. In contrast, the germination proteins were largely biotinylated in germinated spores, although GFP was not. All of the germination proteins in the germinated spore's IM, but not spore core GFP, were largely sensitive to an exogenous protease. These results, coupled with predicted or experimentally determined structural data, indicate that (i) these germination proteins are at least partially and in some cases completely on the outer surface of the spore's IM and (ii) there is significant reorganization of these germination proteins' structure or environment in the IM during spore germination. Spores of Bacillus species can remain dormant for years, yet can return to life within minutes in the process of spore germination followed by outgrowth (1, 2). A number of proteins are specifically involved in spore germination (1-5) including (i) the multiple-nutrient germinant receptors (GRs), each of which recognizes different nutrient germinants and has A, B, and C subunits; (ii) the GerD protein required for rapid GR-dependent germination; (iii) the SpoVA proteins needed for uptake of the spore core's large depot of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) in sporulation and its release in spore germination; and (iv) cortex-lytic enzymes (CLEs) needed to hydrolyze spores' large peptidoglycan (PG) cortex. Many of these proteins, in particular the GRs and the GerD and SpoVA proteins, as well as one CLE (SleB) and the YpeB protein essential for SleB assembly in spores, are in the inner membrane (IM) that surrounds the spore core (6-12), and the GRs and GerD appear to be associated in a small cluster or focus in the IM (7). However, there is no definitive knowledge of the precise topology of any of these proteins in the spore IM other than predicted topology based on the following: (i) knowledge that GRs and GerD and SpoVA proteins are synthesized in the developing spore (1, 2, 4); (ii) the presence or absence of a likely N-terminal signal sequence; (iii) in some cases, a recognition signal for diacylglycerol addition to a cysteine residue near the protein's N terminus (13); and (iv) the facts that the sleB ge...

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Analysis of the Effects of a gerP Mutation on the Germination of Spores of Bacillus subtilis

Cited by 36 publications

References 47 publications

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

Topology and Accessibility of Germination Proteins in the Bacillus subtilis Spore Inner Membrane

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