Clostridioides difficile SpoVAD and SpoVAE interact and are required for DPA uptake into spores

2021 ◽  
Author(s):  
Marko Baloh ◽  
Joseph A. Sorg
Keyword(s):  
Heat Resistance ◽  
Dipicolinic Acid ◽  
Complex Structure ◽  
Variable Number ◽  
Community Settings ◽  
Clostridioides Difficile ◽  
Stage Of Development ◽  
Core Proteins ◽  
Wet Heat ◽  
Considerable Resistance

Clostridioides difficile spores, like the spores from most endospore-forming organisms, are a metabolically dormant stage of development with a complex structure that conveys considerable resistance to environmental conditions, e.g. , wet heat. This resistance is due to the large amount of dipicolinic acid (DPA) that is taken up by the spore core, preventing rotational motion of the core proteins. DPA is synthesized by the mother cell and its packaging into the spore core is mediated by the products of the spoVA operon, which has a variable number of genes, depending on the organism. C. difficile encodes 3 spoVA orthologues, spoVAC, spoVAD, and spoVAE. Prior work has shown that C. difficile SpoVAC is a mechanosensing protein responsible for DPA release from the spore core upon the initiation of germination. However, the roles of SpoVAD and SpoVAE remain unclear in C. difficile . In this study we analyzed the roles of SpoVAD and SpoVAE and found that they are essential for DPA uptake into the spore, similar to SpoVAC. Using split luciferase protein interaction assays we found that these proteins interact, and we propose a model where SpoVAC/SpoVAD/SpoVAE proteins interact at or near the inner spore membrane, and each member of the complex is essential for DPA uptake into the spore core. Importance C. difficile spore heat resistance provides an avenue for it to survive the disinfection protocols in hospital and community settings. The spore heat resistance is mainly the consequence of the high DPA content within the spore core. By elucidating the mechanism by which DPA is taken up by the spore core, this study may provide insight in how to disrupt the spore heat resistance with the aim of making the current disinfection protocols more efficient at preventing the spread of C. difficile in the environment.

scholarly journals Clostridioides difficile SpoVAD and SpoVAE interact and are required for DPA packaging into spores

2021 ◽  
Author(s):  
Marko Baloh ◽  
Joseph A. Sorg
Keyword(s):  
Dipicolinic Acid ◽  
Complex Structure ◽  
Variable Number ◽  
Dormant Stage ◽  
Dry Heat ◽  
Clostridioides Difficile ◽  
Stage Of Development ◽  
Number Of Genes ◽  
Luciferase Protein ◽  
Considerable Resistance

Clostridioides difficile spores, like the spores from most endospore-forming organisms, are a metabolically dormant stage of development with a complex structure that conveys considerable resistance to environmental conditions, e.g., dry heat. This resistance is due to the large amount of dipicolinic acid (DPA) that is packaged into the spore core, thereby replacing the majority of water. DPA is synthesized by the mother cell and its packaging into the spore core is regulated by the spoVA operon that has a variable number of genes, depending on the organism. C. difficile encodes 3 spoVA orthologues, spoVAC, spoVAD, and spoVAE. Prior work has shown that C. difficile SpoVAC is a mechanosensing protein responsible for DPA release from the spore core upon the initiation of germination. However, the roles of SpoVAD and SpoVAE remain unclear in C. difficile. In this study we analyzed the roles of SpoVAD and SpoVAE and found that they are essential for DPA packaging into the spore, similar to SpoVAC. Using split luciferase protein interaction assays we found that these proteins interact, and we propose a model where SpoVAC / SpoVAD / SpoVAE proteins interact at or near the inner spore membrane, and each member of the complex is essential for DPA packaging into the spore

scholarly journals Molecular Physiological Characterization of a High Heat Resistant Spore Forming Bacillus subtilis Food Isolate

2021 ◽  
Vol 9 (3) ◽  
pp. 667
Author(s):  
Zhiwei Tu ◽  
Peter Setlow ◽  
Stanley Brul ◽  
Gertjan Kramer
Keyword(s):  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Dipicolinic Acid ◽  
Laboratory Strain ◽  
High Heat ◽  
High Heat Resistance ◽  
Vegetative Cells ◽  
Heat Resistant ◽  
Food Isolate ◽  
Wet Heat

Bacterial endospores (spores) are among the most resistant living forms on earth. Spores of Bacillus subtilis A163 show extremely high resistance to wet heat compared to spores of laboratory strains. In this study, we found that spores of B. subtilis A163 were indeed very wet heat resistant and released dipicolinic acid (DPA) very slowly during heat treatment. We also determined the proteome of vegetative cells and spores of B. subtilis A163 and the differences in these proteomes from those of the laboratory strain PY79, spores of which are much less heat resistant. This proteomic characterization identified 2011 proteins in spores and 1901 proteins in vegetative cells of B. subtilis A163. Surprisingly, spore morphogenic protein SpoVM had no homologs in B. subtilis A163. Comparing protein expression between these two strains uncovered 108 proteins that were differentially present in spores and 93 proteins differentially present in cells. In addition, five of the seven proteins on an operon in strain A163, which is thought to be primarily responsible for this strain’s spores high heat resistance, were also identified. These findings reveal proteomic differences of the two strains exhibiting different resistance to heat and form a basis for further mechanistic analysis of the high heat resistance of B. subtilis A163 spores.

Atomistic Study of Wet-heat Resistance of Calcium Dipicolinate in the Core of Spores

MRS Advances ◽  
2018 ◽  
Vol 3 (26) ◽  
pp. 1457-1462
Author(s):  
Ankit Mishra ◽  
Pankaj Rajak ◽  
Subodh Tiwari ◽  
Chunyang Sheng ◽  
Aravind Krishnamoorthy ◽  
...  
Keyword(s):  
Heat Resistance ◽  
Dipicolinic Acid ◽  
Bacterial Spores ◽  
Water Molecules ◽  
Confined Water ◽  
Reactive Molecular Dynamics ◽  
Wet Heat ◽  
Experimental Findings ◽  
Core Simulation ◽  
Solid Liquid

ABSTRACTThe extreme heat resistance of dormant bacterial spores strongly depends on the extent of protoplast dehydration and the concentration of dipicolinic acid (DPA) and its associated calcium salts (Ca-DPA) in the spore core. Recent experiments have suggested that this heat resistance depends on the properties of confined water molecules in the hydrated Ca-DPA-rich protoplasm, but atomistic details have not been elucidated. In this study, we used reactive molecular dynamics (RMD) simulations to study the dynamics of water in hydrated DPA and Ca-DPA as a function of temperature. The RMD simulations indicate two distinct solid-liquid and liquid-gel transitions for the spore core. Simulation results reveal monotonically decreasing solid-gel-liquid transition temperatures with increasing hydration. Additional calculations on the specific heat and free energy of water molecules in the spore core further support the higher heat resistance of dehydrated spores. These results provide an insight into the experimental trend of moist-heat resistance of bacterial spores and reconciles previous conflicting experimental findings on the state of water in bacterial spores.

scholarly journals Superdormant Spores of Bacillus Species Have Elevated Wet-Heat Resistance and Temperature Requirements for Heat Activation

2009 ◽  
Vol 191 (18) ◽  
pp. 5584-5591 ◽  
Author(s):  
Sonali Ghosh ◽  
Pengfei Zhang ◽  
Yong-qing Li ◽  
Peter Setlow
Keyword(s):  
Heat Resistance ◽  
Dipicolinic Acid ◽  
Bacillus Species ◽  
Activation Temperature ◽  
New Information ◽  
Temperature Requirements ◽  
Wet Heat ◽  
Dormant Spore ◽  
Heat Activation ◽  
Temperature Optima

ABSTRACT Purified superdormant spores of Bacillus cereus, B. megaterium, and B. subtilis isolated after optimal heat activation of dormant spores and subsequent germination with inosine, d-glucose, or l-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15°C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations.

scholarly journals The Products of the spoVA Operon Are Involved in Dipicolinic Acid Uptake into Developing Spores of Bacillus subtilis

2002 ◽  
Vol 184 (2) ◽  
pp. 584-587 ◽  
Author(s):  
Federico Tovar-Rojo ◽  
Monica Chander ◽  
Barbara Setlow ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Dipicolinic Acid ◽  
Lytic Enzyme ◽  
Acid Uptake ◽  
Wild Type ◽  
Wet Heat

ABSTRACT Bacillus subtilis cells with mutations in the spoVA operon do not complete sporulation. However, a spoVA strain with mutations that remove all three of the spore’s functional nutrient germinant receptors (termed the ger3 mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation. ger3 spoVA and sleB spoVA spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or ger3 spores. These properties of ger3 spoVA and sleB spoVA spores are identical to those of ger3 spoVF and sleB spoVF spores that lack DPA due to deletion of the spoVF operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in ger3 spoVF spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into ger3 spoVA spores. These data indicate that one or more products of the spoVA operon are involved in DPA transport into the developing forespore during sporulation.

scholarly journals Maturation of Released Spores Is Necessary for Acquisition of Full Spore Heat Resistance during Bacillus subtilis Sporulation

2011 ◽  
Vol 77 (19) ◽  
pp. 6746-6754 ◽  
Author(s):  
Jose-Luis Sanchez-Salas ◽  
Barbara Setlow ◽  
Pengfei Zhang ◽  
Yong-qing Li ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Uv Radiation ◽  
Dipicolinic Acid ◽  
Sporulation Medium ◽  
Content Type ◽  
Dry Heat ◽  
Lower Resistance ◽  
Wet Heat ◽  
Spore Maturation

ABSTRACTThe first ∼10% of spores released from sporangia (early spores) duringBacillus subtilissporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ∼24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca2+but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.

scholarly journals Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase

2016 ◽  
Vol 198 (11) ◽  
pp. 1694-1707 ◽  
Author(s):  
M. Lauren Donnelly ◽  
Kelly A. Fimlaid ◽  
Aimee Shen
Keyword(s):  
Clostridium Difficile ◽  
Heat Resistance ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
The United States ◽  
Spore Formation ◽  
Obligate Anaerobe ◽  
Wild Type ◽  
Content Type ◽  
Wet Heat

ABSTRACTThe spore-forming obligate anaerobeClostridium difficileis a leading cause of antibiotic-associated diarrhea around the world. In order forC. difficileto cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. InC. difficile, cortex hydrolysis is necessary for DPA release, whereas inBacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required forC. difficilespore germination by constructing mutations in eitherspoVACordpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively.C. difficilespoVACanddpaABmutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast,B. subtilisspoVACanddpaABmutant spores were unstable. AlthoughC. difficilespoVACanddpaABmutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly,C. difficilespoVACmutant spores were significantly more sensitive to heat treatment thandpaABmutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase controlC. difficilespore resistance and reveal differential requirements for these proteins among theFirmicutes.IMPORTANCEClostridium difficileis a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential forC. difficileto cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid (DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable forC. difficilespore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms.

scholarly journals Phylogenomic analysis of Clostridioides difficile ribotype 106 strains reveals novel genetic islands and emergent phenotypes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bryan Angelo P. Roxas ◽  
Jennifer Lising Roxas ◽  
Rachel Claus-Walker ◽  
Anusha Harishankar ◽  
Asad Mansoor ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Diarrheal Disease ◽  
The United States ◽  
Genomic Islands ◽  
Rapid Expansion ◽  
Phylogenomic Analysis ◽  
Community Settings ◽  
Hamster Model ◽  
Clostridioides Difficile ◽  
Healthcare Associated

AbstractClostridioides difficile infection (CDI) is a major healthcare-associated diarrheal disease. Consistent with trends across the United States, C. difficile RT106 was the second-most prevalent molecular type in our surveillance in Arizona from 2015 to 2018. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains sequenced to date, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed, in any other microbial genome; however, smaller segments were detected in Enterococcus faecium strains. Molecular clock analyses suggested that GI1 was horizontally acquired and sequentially assembled over time. GI1 encodes homologs of VanZ and a SrtB-anchored collagen-binding adhesin, and correspondingly, all tested RT106 strains had increased teicoplanin resistance, and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands are predicted to encode mobile genetic elements as well as virulence factors. Emergent phenotypes associated with these genetic islands may have contributed to the relatively rapid expansion of RT106 in US healthcare and community settings.

Properties of spores of Bacillus subtilis with or without a transposon that decreases spore germination and increases spore wet heat resistance

2021 ◽  
Author(s):  
Yannong Luo ◽  
George Korza ◽  
Angela M. DeMarco ◽  
Oscar P. Kuipers ◽  
Yong‐qing Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Spore Germination ◽  
Wet Heat

scholarly journals Ecology of seed germination and features of ontogeny of floating mat-forming hygrogelophyte Calla palustris (Araceae) under laboratory conditions

2017 ◽  
Vol 25 (4) ◽  
Author(s):  
E. A. Belyakov ◽  
A. G. Lapirov ◽  
O. A. Lebedeva
Keyword(s):  
Physiological Mechanism ◽  
Early Death ◽  
Variable Number ◽  
Cold Stratification ◽  
Seed Productivity ◽  
Laboratory Conditions ◽  
Stage Of Development ◽  
Wide Range ◽  
Collective Fruit ◽  
Floating Mat

This article examines the ecology of germination and the features of ontogenesis of the floating mat-forming hygrogelophyte Calla palustris L. in the territory of some regions in the central part of European Russia and the Republic of Belarus under laboratory conditions. It has been found that in the surveyed territory in the dense ear-like collective fruit of C. palustris, 40.7 ± 6.4 fruits, juicy berries, are formed and the number of seeds produced (actual seed productivity), averages 164.0 ± 89.3. Widely varying data on the number of berries in the collective fruit, as well as a variable number of ovules in them, shows that the seed productivity of C. palustris depends on a whole range of endogenous and exogenous factors, including the effectiveness of pollination of flowers by insects. In laboratory experiments, various storage periods (2 and 12 months) and methods of presowing seed treatment (stratification, drying, ice-freezing) were applied to simulate the ecological conditions of the growth of white alder. It is shown that freshly harvested seeds do not germinate at once, and wet cold stratification makes it possible to achieve maximum values of laboratory germination (from 84.4 to 99.0) and germination energy (from 66.6 to 88.3). Given that the features of germination are indicators of dormancy, it is demonstrated that seeds of C. palustris are in a state of shallow physiological dormancy, conditioned by the physiological mechanism of inhibition. Along with dry storage, wet cold stratification is the main way for seeds to enter a non-dormant state. Such mechanisms are consistent with the climatic features of the regions in which the species grows. It is found that C. palustris seeds, in common with many other species of hygrogelophytes, can float on the water surface for a long time (more than 30 days), spreading with water flow (hydrochoria). Seeds of C. palustris are photosensitive, germination is observed in a wide range of temperatures – from 10–14 to 30 ºС (at constant humidity), type of germination – underground (hypogeal). It is found that ontogeny of individuals of generative origin of C. palustris in the laboratory is terminated (the plants died after passing the juvenile ontogenetic state). The formation of C. palustris seedlings under laboratory conditions lasts 23–25 days and is characterized by the appearance of the main organs of the plant and the anisotropic growth of the shoot axis. At the final stage of development, the seedling is represented by a uniaxial monopodial and anisotropic growing rosette shoot with shortened internodes. The juvenile ontogenetic state in laboratory conditions lasts up to 7 months, after which the plants die off. In the framework of ontomorphogenesis, the stages of ontogenetic development under study (the seedling and the juvenile plant) correspond to the phase of the primary uniaxial rosette shoot. The plant in this period is represented by a uniaxial monopodially growing anchorage shoot. The detection of virgin plants in natural conditions indicates the possibility of their further development from the rudiments of generative origin. The main way of the species reproduction is vegetative, characteristic of most aquatic and semi-aquatic plants. In the course of ontogenesis, progressive features of development such as cotyledon greening and early death of the radicle root have been revealed.

Sign in / Sign up

Export Citation Format

Share Document