Cortex Hydrolysis Research Articles

Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

Identification of Germinants and Expression of Germination Genes in Clostridium perfringens Strains Isolated from Diarrheic Animals.

In this study, we investigated the spore germination phenotype of Clostridium perfringens strains isolated from diarrheic animals (animal strains). The transcripts of germination-specific genes and their protein products were also measured. Our study found the following results: (i) animal strains spores germinated at a slower rate with AK (mixture of L-asparagine and KCl), L-cysteine, or L-lysine, but the extent of germination varied based on strains and germinants used; (ii) none of the amino acids (excluding L-cysteine and L-lysine) were identified as a universal germinant for spores of animal strains; (iii) animal strain spores germinated better at a pH range of 6.0-7.0; (iv) all tested germination-specific genes were expressed in animal strains; the levels of expression of major germinant receptor gene (gerKC) were higher and the cortex hydrolysis machinery genes (cspB and sleC) were lower in animal strains, compared to the food poisoning strain SM101; and (v) the levels of CspB and SleC were significantly lower in spores of animal strains compared to strain SM101, suggesting that these animal strains lack an efficient spore cortex hydrolysis machinery. In summary, our findings suggest that the poor or slow spore germination in C. perfringens animal strains might be due to incomplete spore cortex hydrolysis.

Characterization of a Clostridioides difficile ST-293 isolate from a recurrent infection in Argentina

Clostridioides difficile is an opportunistic spore-forming pathogen responsible for antibiotic-associated diarrhea in humans. C. difficile produces two main toxins: TcdA and TcdB as well as a third toxin named binary toxin (CDT) that is also involved in virulence. The present study aimed at characterizing the C. difficile isolate ALCD3 involved in a relapse episode of nosocomial infection. Molecular characterization showed that isolate ALCD3 belongs to toxinotype 0/v and the MLST analysis demonstrated allelic profile adk:91, atpA:1, dxr:2, glyA: 1, recA:27, sodA: 1 and tpi:1 which corresponds to ST293 (MLST clade: 1). During growth, isolate ALCD3 showed an early increase in the sporulation ratio as well as maximal values of heat resistant forms after 2 days of incubation. Both sporulation kinetics and production of heat resistant forms were faster for isolate ALCD3 than for the reference strain VPI 10463. Germination in the presence of the natural germinant taurocholate was faster for isolate ALCD3 than for strain VPI 10463, which indicates that isolate ALCD3 starts cortex hydrolysis earlier than strain VPI 10463. Furthermore, the co-germinant glycine, induces rapid release of dipicolinic acid (DPA) in isolate ALCD3. These findings indicate that isolate ALCD3 is particularly efficient in both sporulation and germination. The present work represents the first report of the circulation of C. difficile ST293 in Argentina. The ability of isolate ALCD3 to produce toxins and its high sporulation/germination capacity are key features compatible with a microorganism with high dissemination potential and the possibility of inducing recurrent infections.

Proteomic Response of Bacillus subtilis Spores under High Pressure Combined with Moderate Temperature and Random Peptide Mixture LK Treatment.

In this study, a method of Bacillus subtilis spore inactivation under high pressure (P, 200 MPa) combined with moderate temperature (T, 80 °C) and the addition of antimicrobial peptide LK (102 μg/mL) was investigated. Spores presented cortex hydrolysis and inner membrane (IM) damage with an 8.16 log reduction in response to treatment with PT-LK, as observed by phase-contrast and inverted fluorescence microscopy and flow cytometry (FCM) analysis. Furthermore, a tandem mass tag (TMT) quantitative proteomics approach was utilized because Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis data were used. After treatment with PT-LK, 17,017 polypeptides and 3166 proteins were detected from B. subtilis spores. Among them, 78 proteins showed significant differences in abundance between the PT-LK-treated and control groups, with 49 proteins being upregulated and 29 proteins being downregulated in the PT-LK-treated group. Genetic information processing, metabolism, cellular process, and environmental information processing were the main mechanisms of PT-LK-mediated spore inactivation.

High pressure CO2 reduces the wet heat resistance of Bacillus subtilis spores by perturbing the inner membrane

Spores of wild-type Bacillus subtilis PS533 were treated by wet heat at 75 °C for 30 min, and high pressure CO2 (HPCD) at 6.5 MPa and 30 °C or 75 °C for 30 min. The spores were analyzed for wet heat resistance (85 °C, 90 °C, 95 °C) and typical germination events including DPA release and cortex hydrolysis, inner membrane permeability, and germination triggered by nutrient (L-valine and AGFK) or non-nutrient (dodecylamine and high pressure at 150 MPa or 550 MPa) germinants. The results showed that (i) HPCD-treated spores exhibited reduced wet heat resistance compared to the untreated or wet heat-treated spores; (ii) HPCD-treated spores did not undergo typical germination events such as DPA release or cortex hydrolysis compared to normally germinated spores; (iii) HPCD-treated spores released more metal ions and exhibited decreased ability to maintain DPA, indicating that the permeability of inner membrane of HPCD-treated spores was increased; (iv) HPCD-treated spores exhibited reduced germination rate when triggered by L-valine or 150 MPa, but increased germination rate when triggered by dodecylamine or 550 MPa, suggesting that the fluidity of the inner membrane of HPCD-treated spores might be increased. These results indicated that HPCD could reduce the wet heat resistance of spores, and this resistance decrease was probably due to the modification of the inner membrane caused by HPCD. Industrial relevanceThe extremely high wet heat resistance of spores makes them a significant problem in the thermal processing of foods. Thus, it of great interest to develop a process to reduce the wet heat resistance of spores. In this work, we found that HPCD can significantly reduce the wet heat resistance of B. subtilis spores, and this was achieved by perturbing the inner membrane of spores. These results can improve our understanding of the inactivation mechanism of spores by HPCD, and also provide an alternative approach for spore inactivation in foods.

The serine proteases CspA and CspC are essential for germination of spores of Clostridium perfringens SM101 through activating SleC and cortex hydrolysis

Clostridium perfringens SM101 genome encodes three serine proteases (CspA, CspB, and CspC), and genetic evidence indicates that CspB is required for processing of pro-SleC into active SleC, an enzyme essential for degradation of the peptidoglycan cortex during spore germination. In this study, the expression of cspA and cspC, as well as the germination and colony formation by spores of cspAC and cspC mutants of strain SM101, were assessed. We demonstrated that 1) the cspA and cspC genes were expressed as a bicistronic operon only during sporulation in the mother cell compartment of SM101; 2) both cspAC and cspC mutant spores were unable to germinate significantly with either KCl, l-glutamine, brain heart infusion (BHI) broth, or a 1:1 chelate of Ca2+ and dipicolinic acid (DPA); 3) consistent with germination results, both cspAC and cspC mutant spores were defective in normal DPA release; 4) the colony formation by cspAC and cspC mutant spores was ~106-fold lower than that of wild-type spores, although decoated mutant spores yielded wild-type level colony formation on plates containing lysozyme; 5) no processing of inactive pro-SleC into active SleC was observed in cspAC and cspC mutant spores during germination; and finally, 6) the defects in germination, DPA release, colony formation and SleC processing in cspAC and cspC mutant spores were complemented by the wild-type cspA-cspC operon. Collectively, these results indicate that both CspA and CspC are essential for C. perfringens spore germination through activating SleC and inducing cortex hydrolysis.

Novel cortex lytic enzymes in Bacillus megaterium QM B1551 spores.

Present models for spore germination in Bacillus species include a requirement for either the SleB or CwlJ cortex lytic enzymes to efficiently depolymerise the spore cortex. Previous work has demonstrated that B. megaterium spores may differ to other species in this regard, since sleB cwlJ null mutant spores complemented with the gene in trans for the non-peptidoglycan lysin YpeB can efficiently degrade the cortex. Here, we identify two novel cortex lytic enzymes, encoded at the BMQ_2391 and BMQ_3234 loci, which are essential for cortex hydrolysis in the absence of SleB and CwlJ. Ellipsoid localisation microscopy places the BMQ_3234 protein within the inner-spore coat, a region of the spore that is populated by other cortex lytic enzymes. The findings reinforce the idea that there is a degree of variation in mechanisms of cortex hydrolysis across the Bacillales, raising potential implications for environmental decontamination strategies based upon targeted inactivation of components of the spore germination apparatus.

Analysis of the Germination of Individual Clostridium sporogenes Spores with and without Germinant Receptors and Cortex-Lytic Enzymes.

The Gram-positive spore-forming anaerobe Clostridium sporogenes is a significant cause of food spoilage, and it is also used as a surrogate for C. botulinum spores for testing the efficacy of commercial sterilization. C. sporogenes spores have also been proposed as a vector to deliver drugs to tumor cells for cancer treatments. Such an application of C. sporogenes spores requires their germination and return to life. In this study, Raman spectroscopy and differential interference contrast (DIC) microscopy were used to analyze the germination kinetics of multiple individual C. sporogenes wild-type and germination mutant spores. Most individual C. sporogenes spores germinated with L-alanine began slow leakage of ∼5% of their large Ca-dipicolinic acid (CaDPA) depot at T1, all transitioned to rapid CaDPA release at Tlag1, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlys. T1, Tlag1, Trelease, and Tlys times for individual spores were heterogeneous, but ΔTrelease (Trelease – Tlag1) periods were relatively constant. However, variability in T1 (or Tlag1) times appeared to be the major reason for the heterogeneity between individual spores in their germination times. After Trelease, some spores also displayed another lag in rate of change in DIC image intensity before the start of a second obvious DIC image intensity decline of 25–30% at Tlag2 prior to Tlys. This has not been seen with spores of other species. Almost all C. sporogenes spores lacking the cortex-lytic enzyme (CLE) CwlJ spores exhibited a Tlag2 in L-alanine germination. Sublethal heat treatment potentiated C. sporogenes spore germination with L-alanine, primarily by shortening T1 times. Spores without the CLEs SleB or CwlJ exhibited greatly slowed germination with L-alanine, but spores lacking all germinant receptor proteins did not germinate with L-alanine. The absence of these various germination proteins also decreased but did not abolish germination with the non-GR-dependent germinants dodecylamine and CaDPA, but spores without CwlJ exhibited no germination with CaDPA. Finally, C. sporogenes spores displayed commitment in germination, but memory in GR-dependent germination was small, and less than the memory in Bacillus spore germination.

Germination of Spores of the Orders Bacillales and Clostridiales

Dormant Bacillales and Clostridiales spores begin to grow when small molecules (germinants) trigger germination, potentially leading to food spoilage or disease. Germination-specific proteins sense germinants, transport small molecules, and hydrolyze specific bonds in cortex peptidoglycan and specific proteins. Major events in germination include (a) germinant sensing; (b) commitment to germinate; (c) release of spores' depot of dipicolinic acid (DPA); (d) hydrolysis of spores' peptidoglycan cortex; and (e) spore core swelling and water uptake, cell wall peptidoglycan remodeling, and restoration of core protein and inner spore membrane lipid mobility. Germination is similar between Bacillales and Clostridiales, but some species differ in how germinants are sensed and how cortex hydrolysis and DPA release are triggered. Despite detailed knowledge of the proteins and signal transduction pathways involved in germination, precisely what some germination proteins do and how they do it remain unclear.

Proteins YlaJ and YhcN contribute to the efficiency of spore germination in Bacillus subtilis.

The YlaJ and YhcN spore lipoproteins of Bacillus subtilis contain a common domain, and are of unknown function. Homologues of YlaJ or YhcN are widespread in Bacilli and are also encoded in those Clostridia that use cortex lytic enzymes SleB and CwlJ for cortex hydrolysis during germination. In B. subtilis, we report that single and double mutants lacking YlaJ and/or YhcN show a reduced rate of spore germination in L-alanine, with a delay in loss of heat resistance, release of dipicolinic acid and OD fall. If B. subtilis spores lack the cortex lytic enzyme CwlJ, spore cortex degradation and subsequent outgrowth to form colonies is strictly dependent on the other cortex lytic enzyme SleB, allowing a test of SleB function; in a cwlJ mutant background, the combined loss of both ylaJ and yhcN genes resulted in a spore population in which only 20% of spores germinated and outgrew to form colonies, suggesting that SleB activity is compromised. YlaJ and YhcN have a role in germination that is not yet well defined, but these proteins are likely to contribute, directly or indirectly, to early events in germination, including effective SleB function.

Commentary: Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination.

Dormant bacterial spores can rapidly revive and resume a vegetative life form once nutrients become available (Stragier and Losick, 1996; Setlow, 2003). The earliest revival event, termed germination, holds the key to understanding this rapid conversion from a dormant into an active cell. During germination, the spore undergoes release of dipicolinic acid (DPA), rehydration, cortex hydrolysis, and coat disassembly. This phase is accompanied by transition from a phase-bright spore to a phase-dark cell, as manifested by light microscopy (Setlow, 2003, 2013; Moir, 2006; Figure ​Figure1A).1A). Germination is traditionally considered to occur without the need for any macromolecule synthesis (Steinberg et al., 1965; Vinter, 1970; Setlow, 2003, 2013; Moir, 2006); however, our recent results challenge this view, as we demonstrated that translation occurs during germination and is required for its execution (Sinai et al., 2015).

The crystal structure of Clostridium perfringens SleM, a muramidase involved in cortical hydrolysis during spore germination.

Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination. SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM. Here, we present the crystal structure of the C. perfringens S40 SleM protein at 1.8 Å. SleM comprises an N-terminal catalytic domain that adopts an irregular α/β-barrel fold that is common to GH25 family lysozymes, plus a C-terminal fibronectin type III domain. The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution. A truncated form of SleM that lacks the FnIII domain shows reduced activity against spore sacculi indicating that this domain may have a role in facilitating the position of substrate with respect to the enzyme's active site. Proteins 2016; 84:1681-1689. © 2016 Wiley Periodicals, Inc.

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

ABSTRACTThe spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to 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. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant 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. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes.IMPORTANCE Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to 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 for C. difficile spore 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.

Involvement of Coat Proteins in Bacillus subtilis Spore Germination in High-Salinity Environments.

The germination of spore-forming bacteria in high-salinity environments is of applied interest for food microbiology and soil ecology. It has previously been shown that high salt concentrations detrimentally affect Bacillus subtilis spore germination, rendering this process slower and less efficient. The mechanistic details of these salt effects, however, remained obscure. Since initiation of nutrient germination first requires germinant passage through the spores' protective integuments, the aim of this study was to elucidate the role of the proteinaceous spore coat in germination in high-salinity environments. Spores lacking major layers of the coat due to chemical decoating or mutation germinated much worse in the presence of NaCl than untreated wild-type spores at comparable salinities. However, the absence of the crust, the absence of some individual nonmorphogenetic proteins, and the absence of either CwlJ or SleB had no or little effect on germination in high-salinity environments. Although the germination of spores lacking GerP (which is assumed to facilitate germinant flow through the coat) was generally less efficient than the germination of wild-type spores, the presence of up to 2.4 M NaCl enhanced the germination of these mutant spores. Interestingly, nutrient-independent germination by high pressure was also inhibited by NaCl. Taken together, these results suggest that (i) the coat has a protective function during germination in high-salinity environments; (ii) germination inhibition by NaCl is probably not exerted at the level of cortex hydrolysis, germinant accessibility, or germinant-receptor binding; and (iii) the most likely germination processes to be inhibited by NaCl are ion, Ca(2+)-dipicolinic acid, and water fluxes.

Sporicidal Activities of Various Surfactant Components against Bacillus subtilis Spores

The sporicidal activities against Bacillus subtilis spores of surfactant components with hydrophilic and hydrophobic properties that can lead to the denaturation of various proteins comprising the spore structure were investigated. The reduction in spore numbers by each of the surfactant components bornyl acetate, geranyl acetate, pinene, p-cymene, camphene, citral, 2,3-dihydrobenzofuran, polylysine, and thiamine dilaurylsulfate at 1% was estimated at 1 to 2 log CFU/ml. The average hydrophile-lipophile balance value of surfactants with sporicidal activity causing a reduction of 1 to 2 log CFU/ml was 9.3, with a range from 6.7 to 15.8, which is similar to the values of various chemical surfactants of 9.6 to 16.7. The results also showed that the surfactants that were hydrophobic were more effective than those that were hydrophilic in killing B. subtilis spores. Furthermore, the sporicidal effect of surfactants like geranyl acetate and γ-terpinene was significantly enhanced in the presence of a germinant, because l-alanine and synergistic cofactors (e.g., K+ ions) trigger cortex hydrolysis in spores.

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

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 ∼15% concentration of a chelate of Ca(2+) and dipicolinic acid (CaDPA) at a heterogeneous time T1, rapidly released CaDPA at Tlag, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlysis. T1 and Tlag values for individual spores were heterogeneous, but ΔTrelease periods (Trelease - Tlag) 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 Trelease, 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. Spores 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.

Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination.

Bacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis during B. subtilis spore germination. Here, we investigated the timing of DPA release and cortex hydrolysis during Clostridium difficile spore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor, cspC, or the cortex hydrolase, sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release during C. difficile spore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediated C. difficile spore germination proceeds through a novel germination pathway. Clostridium difficile infects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease. C. difficile spore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to the Bacillus subtilis germinant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism and C. difficile. We found that C. difficile spore germination proceeds through a novel pathway.

Location and stoichiometry of the protease CspB and the cortex-lytic enzyme SleC in Clostridium perfringens spores

The protease CspB and the cortex-lytic enzyme SleC are essential for peptoglycan cortex hydrolysis during germination of spores of the Clostridium perfringens food poisoning isolate SM101. In this study, Western blot analyses were used to demonstrate that CspB and SleC are present exclusively in the C. perfringens SM101 spore coat layer fraction and absent in the lysate from decoated spores and from the purified inner spore membrane. These results indicate why decoating treatments greatly reduce both germination and apparent viability of C. perfringens spores in the absence of an exogenous lytic enzyme. In addition, quantitative Western blot analyses showed that there are approximately 2000 and 130,000 molecules of CspB and pro-SleC, respectively, per C. perfringens SM101 spore, consistent with CspB's role in acting catalytically on pro-SleC to convert this zymogen to the active enzyme.

Carvacrol suppresses high pressure high temperature inactivation of Bacillus cereus spores

The inactivation of bacterial spores generally proceeds faster and at lower temperatures when heat treatments are conducted under high pressure, and high pressure high temperature (HPHT) processing is, therefore, receiving an increased interest from food processors. However, the mechanisms of spore inactivation by HPHT treatment are poorly understood, particularly at moderately elevated temperature. In the current work, we studied inactivation of the spores of Bacillus cereus F4430/73 by HPHT treatment for 5min at 600MPa in the temperature range of 50–100°C, using temperature increments of 5°C. Additionally, we investigated the effect of the natural antimicrobial carvacrol on spore germination and inactivation under these conditions. Spore inactivation by HPHT was less than about 1logunit at 50 to 70°C, but gradually increased at higher temperatures up to about 5logunits at 100°C. DPA release and loss of spore refractility in the spore population were higher at moderate (≤65°C) than at high (≥70°C) treatment temperatures, and we propose that moderate conditions induced the normal physiological pathway of spore germination resulting in fully hydrated spores, while at higher temperatures this pathway was suppressed and replaced by another mechanism of pressure-induced dipicolinic acid (DPA) release that results only in partial spore rehydration, probably because spore cortex hydrolysis is inhibited. Carvacrol strongly suppressed DPA release and spore rehydration during HPHT treatment at ≤65°C and also partly inhibited DPA release at ≥65°C. Concomitantly, HPHT spore inactivation was reduced by carvacrol at 65–90°C but unaffected at 95–100°C.

Role of YpeB in cortex hydrolysis during germination of Bacillus anthracis spores.

The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis, and YpeB, a protein necessary for SleB stability and function. The results indicate that ΔsleB and ΔypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup.