scholarly journals Kinetics of Germination of Wet-Heat-Treated Individual Spores of Bacillus Species, Monitored by Raman Spectroscopy and Differential Interference Contrast Microscopy

2011 ◽  
Vol 77 (10) ◽  
pp. 3368-3379 ◽  
Author(s):  
Guiwen Wang ◽  
Pengfei Zhang ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
Heat Treatment ◽  
Raman Spectroscopy ◽  
Spore Germination ◽  
Great Majority ◽  
Lytic Enzyme ◽  
Differential Interference Contrast ◽  
Content Type ◽  
Heat Treated ◽  
Wet Heat ◽  
Kinetics Of

ABSTRACTRaman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the kinetics of nutrient and nonnutrient germination of multiple individual untreated and wet-heat-treated spores ofBacillus cereusandBacillus megaterium, as well as of several isogenicBacillus subtilisstrains. Major conclusions from this work were as follows. (i) More than 90% of these spores were nonculturable but retained their 1:1 chelate of Ca2+and dipicolinic acid (CaDPA) when incubated in water at 80 to 95°C for 5 to 30 min. (ii) Wet-heat treatment significantly increased the time,Tlag, at which spores began release of the great majority of their CaDPA during the germination ofB. subtilisspores with different nutrient germinants and also increased the variability ofTlagvalues. (iii) The time period, ΔTrelease, betweenTlagand the time,Trelease, at which a spore germinating with nutrients completed the release of the great majority of its CaDPA, was also increased in wet-heat-treated spores. (iv) Wet-heat-treated spores germinating with nutrients had higher values ofIrelease, the intensity of a spore's DIC image atTrelease, than did untreated spores and had much longer time periods, ΔTlys, for the reduction inIreleaseintensities to the basal value due to hydrolysis of the spore's peptidoglycan cortex, probably due at least in part to damage to the cortex-lytic enzyme CwlJ. (v) Increases inTlagand ΔTreleasewere also observed when wet-heat-treatedB. subtilisspores were germinated with the nonnutrient dodecylamine, while the change inIreleasewas less significant. (vi) The effects of wet-heat treatment on nutrient germination ofB. cereusandB. megateriumspores were generally similar to those onB. subtilisspores. These results indicate that (i) some proteins important in spore germination are damaged by wet-heat treatment, (ii) the cortex-lytic enzyme CwlJ is one germination protein damaged by wet heat, and (iii) the CaDPA release process itself seems likely to be the target of wet-heat damage which has the greatest effect on spore germination.

scholarly journals Germination, Outgrowth, and Vegetative-Growth Kinetics of Dry-Heat-Treated Individual Spores ofBacillusSpecies

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Lin He ◽  
Zhan Chen ◽  
Shiwei Wang ◽  
Muying Wu ◽  
Peter Setlow ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Spore Germination ◽  
Differential Interference Contrast Microscopy ◽  
Content Type ◽  
Dry Heat ◽  
Heat Treated ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Dry Heat Treatment ◽  
Kinetics Of

ABSTRACTDNA damage kills dry-heated spores ofBacillus subtilis, but dry-heat-treatment effects on spore germination and outgrowth have not been studied. This is important, since if dry-heat-killed spores germinate and undergo outgrowth, toxic proteins could be synthesized. Here, Raman spectroscopy and differential interference contrast microscopy were used to study germination and outgrowth of individual dry-heat-treatedB. subtilisandBacillus megateriumspores. The major findings in this work were as follows: (i) spores dry-heat-treated at 140°C for 20 min lost nearly all viability but retained their Ca2+-dipicolinic acid (CaDPA) depot; (ii) in most cases, dry-heat treatment increased the average times and variability of all major germination events inB. subtilisspore germination with nutrient germinants or CaDPA, and in one nutrient germination event withB. megateriumspores; (iii)B. subtilisspore germination with dodecylamine, which activates the spore CaDPA release channel, was unaffected by dry-heat treatment; (iv) these results indicate that dry-heat treatment likely damages spore proteins important in nutrient germinant recognition and cortex peptidoglycan hydrolysis, but not CaDPA release itself; and (v) analysis of single spores incubated on nutrient-rich agar showed that while dry-heat-treated spores that are dead can complete germination, they cannot proceed into outgrowth and thus not to vegetative growth. The results of this study provide new information on the effects of dry heat on bacterial spores and indicate that dry-heat sterilization regimens should produce spores that cannot outgrow and thus cannot synthesize potentially dangerous proteins.IMPORTANCEMuch research has shown that high-temperature dry heat is a promising means for the inactivation of spores on medical devices and spacecraft decontamination. Dry heat is known to killBacillus subtilisspores by DNA damage. However, knowledge about the effects of dry-heat treatment on spore germination and outgrowth is limited, especially at the single spore level. In the current work, Raman spectroscopy and differential interference contrast microscopy were used to analyze CaDPA levels in and kinetics of nutrient- and non-nutrient germination of multiple individual dry-heat-treatedB. subtilisandBacillus megateriumspores that were largely dead. The outgrowth and subsequent cell division of these germinated but dead dry-heat-treated spores were also examined. The knowledge obtained in this study will help understand the effects of dry heat on spores both on Earth and in space, and indicates that dry heat can be safely used for sterilization purposes.

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

2015 ◽  
Vol 197 (14) ◽  
pp. 2361-2373 ◽  
Author(s):  
Shiwei Wang ◽  
Aimee Shen ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
Raman Spectroscopy ◽  
Gastrointestinal Tract ◽  
Clostridium Difficile ◽  
Spore Germination ◽  
Bile Salts ◽  
Differential Interference Contrast ◽  
Gram Positive ◽  
Content Type ◽  
Kinetics Of ◽  
Dic Microscopy

ABSTRACTThe Gram-positive spore-forming anaerobeClostridium difficileis a leading cause of nosocomial diarrhea. Spores ofC. difficileinitiate infection when triggered to germinate by bile salts in the gastrointestinal tract. We analyzed germination kinetics of individualC. difficilespores using Raman spectroscopy and differential interference contrast (DIC) microscopy. Similar toBacillusspores, individualC. difficilespores germinating with taurocholate plus glycine began slow leakage of a ∼15% concentration of a chelate of Ca2+and dipicolinic acid (CaDPA) at a heterogeneous timeT1, rapidly released CaDPA atTlag, completed CaDPA release atTrelease, and finished peptidoglycan cortex hydrolysis atTlysis.T1andTlagvalues for individual spores were heterogeneous, but ΔTreleaseperiods (Trelease−Tlag) were relatively constant. In contrast toBacillusspores, heat treatment did not stimulate spore germination in the twoC. difficilestrains tested.C. difficilespores 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. difficilespores did not germinate with CaDPA, in contrast toB. subtilisandC. perfringensspores. However, the detergent dodecylamine inducedC. difficilespore germination, and rates were increased by spore coat removal although cortex hydrolysis did not followTrelease, in contrast withB. subtilis.C. difficilespores lacking the cortex-lytic enzyme, SleC, germinated extremely poorly, and cortex hydrolysis was not observed in the fewsleCspores that partially germinated. Overall, these findings indicate thatC. difficileandB. subtilisspore germination exhibit key differences.IMPORTANCESpores of the Gram-positive anaerobeClostridium difficileare responsible for initiating infection by this important nosocomial pathogen. When exposed to germinants such as bile salts,C. difficilespores 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 individualC. difficilespores, 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 inC. difficilespores differs from that inBacillusspores and provide new insights intoC. difficilespore germination.

scholarly journals Monitoring the Wet-Heat Inactivation Dynamics of Single Spores of Bacillus Species by Using Raman Tweezers, Differential Interference Contrast Microscopy, and Nucleic Acid Dye Fluorescence Microscopy

2011 ◽  
Vol 77 (14) ◽  
pp. 4754-4769 ◽  
Author(s):  
Pengfei Zhang ◽  
Lingbo Kong ◽  
Guiwen Wang ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
Heat Treatment ◽  
Nucleic Acid ◽  
Fluorescence Microscopy ◽  
Fluorescence Intensity ◽  
Dipicolinic Acid ◽  
Heat Inactivation ◽  
Bacillus Species ◽  
Differential Interference Contrast ◽  
Content Type ◽  
Wet Heat

ABSTRACTDynamic processes during wet-heat treatment of individual spores ofBacillus cereus,Bacillus megaterium, andBacillus subtilisat 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca2+with dipicolinic acid (CaDPA) was released rapidly at a highly variable timeTlag, the levels of spore nucleic acids remained nearly unchanged, and theTlagtimes for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ∼50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time beforeTlagand reached maximum at a time slightly later thanTrelease. However, the fluorescence intensities of wet-heat-inactivated spores were ∼15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment.

scholarly journals Studying of 2D Titanium Carbide Structure by Raman Spectroscopy after Heat Treatment in Argon and Hydrogen Atmospheres

2017 ◽  
Vol 19 (2) ◽  
pp. 181 ◽  
Author(s):  
Olzhas Kaipoldayev ◽  
Ye. Mukhametkarimov ◽  
Renata Nemkaeva ◽  
G. Baigarinova ◽  
Madi Aitzhanov ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Raman Spectroscopy ◽  
Titanium Carbide ◽  
Functional Groups ◽  
Optimal Regime ◽  
Heat Treated ◽  
Initial Oxygen Content ◽  
Initial Oxygen ◽  
Tio2 Rutile ◽  
Total Disappearance

Herein we show the effect of heat treatment of two dimensional layered titanium carbide structure (Ti3C2Tx), so called MXene. As prepared MXene has functional groups -OH, -F, -Cl. In order to remove the functional groups we heat treated the MXene in Ar (with 0.01% O2) and H2 (with 0.01% H2O) atmospheres. We discovered the significant decrease in the amount of functional groups (-F and -Cl) and increase in the -O content, which refers to the oxidation of the material. Also we determined the optimal regime for Raman spectroscopy in order to avoid any changes in the structure of the material. We revealed that titanium carbide changes its structure at 700 °C and 900 °C into two different titanium dioxide modifications like rutile and anatase in Ar (with 0.01% O2) atmosphere. Also there are small changes occurred in Ti3C2Tx structure and formation of amorphous carbon after 700 °C treatment in H2 (with 0.01% H2O) atmosphere and formation of TiO2 (rutile) at 900 °C. Energydispersive X-ray spectroscopy (EDX) revealed the reduction of functional groups at 700 °C in both atmospheres and total disappearance of –F and –Cl and increasing the oxygen at 900 °C. The huge increase of oxygen by atomic percent, can be explained by the initial oxygen content in argon and hydrogen gases.

Study of Raman Spectroscopy on Graphene Phase from Heat Treatment of Coconut (Cocus nucifera) Shell

2015 ◽  
Vol 827 ◽  
pp. 290-293 ◽  
Author(s):  
Fandi Angga Prasetya ◽  
Muhammad Nasrullah ◽  
Ananda Yogi Nugraheni ◽  
Darminto
Keyword(s):  
Heat Treatment ◽  
Raman Spectroscopy ◽  
Atmospheric Condition ◽  
Peak Position ◽  
Special Treatment ◽  
Distilled Water ◽  
X Ray ◽  
Heat Treated ◽  
Reduced Graphene ◽  
Two Samples

Coconut (Cocus Nucifera) shell as the main ingredient in this research has been heat-treated at temperature of 1000°C in atmospheric condition aiming to obtain the expected phase of graphene. After heat treatment, an additional special treatment was given, where sample was then rinsed with distilled water. Furthermore, the heated coconut shell was characterized by Raman Spectroscopy (785 nm) and X-ray diffractometry. Based on the treatment and characterization conducted, all samples were likely to contain reduced graphene oxide (RGO) phase.The XRD data have supported the existence of RGO with the diffraction peak position (2q) at 25o and 45o. Evidence is also given by the result of Raman Spectroscopy which produces peaks (denoted by D and G bands) located at wave numbers of 1300 cm-1 and 1590 cm-1. The value of the ratio ID/IG of the two samples in the figures are 2.6 and 2.51 (matched with ratio ID/IG of RGO). The ID/IG ratio of sample which was rinsed by distilled water is higher that those without rinsing treatment.

scholarly journals Suboptimal Bacillus licheniformis and Bacillus weihenstephanensis Spore Incubation Conditions Increase Heterogeneity of Spore Outgrowth Time

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
C. Trunet ◽  
N. Mtimet ◽  
A.-G. Mathot ◽  
F. Postollec ◽  
I. Leguerinel ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Flow Cytometry ◽  
Bacillus Licheniformis ◽  
Model Parameters ◽  
Vegetative Cells ◽  
Content Type ◽  
Bacillus Weihenstephanensis ◽  
Spore Outgrowth ◽  
Growth Limits ◽  
Kinetics Of

ABSTRACT Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges of 4.7 to 7.4 and temperatures of 10°C to 37°C for B. weihenstephanensis and 18°C to 59°C for B. licheniformis. Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, a lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C for 12 min or at 95°C for 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite the fact that they both led to a 10-fold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth 1.2-fold and lowered the proportion of spores able to germinate and outgrow 1.4-fold. Interestingly, we showed by proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 than in those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH. IMPORTANCE Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of Bacillus spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sublethal heat treatment, a common situation in food processing and a concern for food preservation and safety.

Corrosion behavior of heat-treated and cryorolled Al 5052 alloys in different chloride ion concentrations

2020 ◽  
Vol 67 (1) ◽  
pp. 7-15 ◽  
Author(s):  
Anasyida Abu Seman ◽  
Ji Kit Chan ◽  
Muhammad Anas Norazman ◽  
Zuhailawati Hussain ◽  
Dhindaw Brij ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Corrosion Behaviour ◽  
Solution Treatment ◽  
Chloride Ion ◽  
Optical Microscope ◽  
Al Alloy ◽  
Content Type ◽  
Ion Concentrations ◽  
Heat Treated ◽  
Chloride Environment

Purpose This paper aims to investigate the corrosion behaviour of heat-treated and cryorolled Al 5052 alloys in different Cl− ion concentrations. Design/methodology/approach NaCl solutions with concentrations of 0, 0.5, 3.5 and 5.5 per cent were selected. Samples were subjected to pre-heat treatment (annealing at 300 °C and solution treatment at 540 °C) and cryorolling up to 30 per cent reduction before undergoing corrosion tests. The corrosion behaviour of the samples was then investigated by potentiodynamic polarization. The microstructure of the corroded samples was evaluated under an optical microscope, and the percentages of pits on their surfaces were calculated. Findings The cryorolled samples had a lower corrosion rate than the samples that were not cryorolled. The cryorolled sample that underwent solution treatment showed the highest corrosion resistance among all the samples tested. Practical implications The commercial impact of the study is the possibility of using the cryorolled Al alloy in various ion chloride environment. Originality/value The obtained results help in understanding the corrosion behaviour of cryorolled samples under different heat treatment conditions.

Failure analysis of heat treated HSLA wheel bolt steels

2010 ◽  
Vol 6 (3) ◽  
pp. 373-382
Author(s):  
Ali Nazari ◽  
Shadi Riahi
Keyword(s):  
Heat Treatment ◽  
Tensile Strength ◽  
Failure Analysis ◽  
Heat Treating ◽  
Boron Steel ◽  
Content Type ◽  
Heat Treated ◽  
Before And After ◽  
Molybdenum Steel ◽  
Optimum Heat

PurposeThe aims of this study is to analyze failure of two types of high‐strength low‐alloy (HSLA) steels which are used in wheel bolts 10.9 grade, boron steel and chromium‐molybdenum steel, before and after heat treatment.Design/methodology/approachThe optimum heat treatment to obtain the best tensile behavior was determined and Charpy impact and Rockwell hardness tests were performed on the two steel types before and after the optimum heat treating.FindingsFractographic studies show a ductile fracture for heat‐treated boron steel while indicate a semi‐brittle fracture for heat‐treated chromium‐molybdenum steel. Formation of a small boron carbide amount during heat treating of boron steel results in increment the bolt's tensile strength while the ductility did not changed significantly. In the other hand, formation of chromium and molybdenum carbides during heat treating of chromium‐molybdenum steel increased the bolt's tensile strength with a considerable reduction in the final ductility.Originality/valueThis paper evaluates failure analysis of HSLA wheel bolt steels and compares their microstructure before and after the loading regime.

Effect of post-heat treatment cooling on microstructure and mechanical properties of selective laser melting manufactured austenitic 316L stainless steel

2020 ◽  
Vol 26 (10) ◽  
pp. 1739-1749
Author(s):  
Saad Waqar ◽  
Jiangwei Liu ◽  
Qidong Sun ◽  
Kai Guo ◽  
Jie Sun
Keyword(s):  
Mechanical Properties ◽  
Heat Treatment ◽  
Grain Size ◽  
316L Stainless Steel ◽  
Laser Melting ◽  
Content Type ◽  
Post Annealing ◽  
Cooling Conditions ◽  
Microstructure And Mechanical Properties ◽  
Heat Treated

Purpose This paper aims to investigate the influence of different post-annealing cooling conditions, i.e. furnace cooling (heat treatment (HT) 1 – slow cooling) and air cooling (HT 2 – fast cooling), on the microstructure and mechanical properties of selective laser melting (SLM) built austenitic 316L stainless steel (SS). Design/methodology/approach Three sets of 316L SS samples were fabricated using a machine standard scanning strategy. Each set consists of three tensile samples and a cubic sample for microstructural investigations. Two sets were subsequently subjected to annealing HT with different cooling conditions, i.e. HT 1 and HT 2, whereas one set was used in the as-built (AB) condition. The standard metallographic techniques of X-ray diffraction, scanning electron microscopy and electron back-scattered diffraction were used to investigate the microstructural variations induced by different cooling conditions. The resultant changes in mechanical properties were also investigated. Findings The phase change of SLM fabricated 316L was observed to be independent of the investigated cooling conditions and all samples consist of austenite phase only. Both HT 1 and HT 2 lead to dissolved characteristic melt pools of SLM. Noticeable increase in grain size of HT 1 and HT 2 samples was also observed. Compared with AB samples, the grain size of HT 1 and HT 2 was increased by 12.5% and 50%, respectively. A decreased hardness and strength, along with an increased ductility was also observed for HT 2 samples compared with HT 1 and AB samples. Originality/value From previous studies, it has been noticed that most investigations on HT of SLM fabricated 316L were mainly focused on the HT temperature or holding time. However, the post-HT cooling rate is also an equally important factor in deciding the microstructure and mechanical properties of heat-treated components. Therefore, this paper investigates the influence of different post-annealing cooling conditions on microstructure and mechanical properties of SLM fabricated 316L components. This study provides a foundation for considering the post-HT cooling rate as an influential parameter that controls the properties of heat-treated SLM components.

scholarly journals Clostridium difficileLipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Oscar R. Diaz ◽  
Cameron V. Sayer ◽  
David L. Popham ◽  
Aimee Shen
Keyword(s):  
Clostridium Difficile ◽  
Spore Germination ◽  
Protective Layer ◽  
Thick Layer ◽  
Lytic Enzyme ◽  
Gram Positive ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
Biochemical Analyses

ABSTRACTClostridium difficile, also known asClostridioides difficile, is a Gram-positive, spore-forming bacterium that is a leading cause of antibiotic-associated diarrhea.C. difficileinfections begin when its metabolically dormant spores germinate to form toxin-producing vegetative cells. Successful spore germination depends on the degradation of the cortex, a thick layer of modified peptidoglycan that maintains dormancy. Cortex degradation is mediated by the SleC cortex lytic enzyme, which is thought to recognize the cortex-specific modification muramic-δ-lactam.C. difficilecortex degradation also depends on thePeptostreptococcaceae-specific lipoprotein GerS for unknown reasons. In this study, we tested whether GerS regulates production of muramic-δ-lactam and thus controls the ability of SleC to recognize its cortex substrate. By comparing the muropeptide profiles of ΔgerSspores to those of spores lacking either CwlD or PdaA, both of which mediate cortex modification inBacillus subtilis, we determined thatC. difficileGerS, CwlD, and PdaA are all required to generate muramic-δ-lactam. Both GerS and CwlD were needed to cleave the peptide side chains from N-acetylmuramic acid, suggesting that these two factors act in concert. Consistent with this hypothesis, biochemical analyses revealed that GerS and CwlD directly interact and that CwlD modulates GerS incorporation into mature spores. Since ΔgerS, ΔcwlD, and ΔpdaAspores exhibited equivalent germination defects, our results indicate thatC. difficilespore germination depends on cortex-specific modifications, reveal GerS as a novel regulator of these processes, and highlight additional differences in the regulation of spore germination inC. difficilerelative toB. subtilisand other spore-forming organisms.IMPORTANCEThe Gram-positive, spore-forming bacteriumClostridium difficileis a leading cause of antibiotic-associated diarrhea. BecauseC. difficileis an obligate anaerobe, its aerotolerant spores are essential for transmitting disease, and their germination into toxin-producing cells is necessary for causing disease. Spore germination requires the removal of the cortex, a thick layer of modified peptidoglycan that maintains spore dormancy. Cortex degradation is mediated by the SleC hydrolase, which is thought to recognize cortex-specific modifications. Cortex degradation also requires the GerS lipoprotein for unknown reasons. In our study, we tested whether GerS is required to generate cortex-specific modifications by comparing the cortex composition of ΔgerSspores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwlD and PdaA. These analyses revealed that GerS, CwlD, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in ΔgerS, ΔcwlD, and ΔpdaAmutants resulted in spore germination and heat resistance defects, the SleC cortex lytic enzyme depends on cortex-specific modifications to efficiently degrade this protective layer. Our results further indicate that GerS and CwlD are mutually required for removing peptide chains from spore peptidoglycan and revealed a novel interaction between these proteins. Thus, our findings provide new mechanistic insight intoC. difficilespore germination.

Sign in / Sign up

Export Citation Format

Share Document