The Clostridium perfringens Germinant Receptor Protein GerKC Is Located in the Spore Inner Membrane and Is Crucial for Spore Germination

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

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Evaluation of the Microbial Quality of Tajik Sambusa and Control of Clostridium perfringens Germination and Outgrowth by Buffered Sodium Citrate and Potassium Lactate†

Journal of Food Protection ◽

10.4315/0362-028x-71.1.77 ◽

2008 ◽

Vol 71 (1) ◽

pp. 77-82 ◽

Cited By ~ 4

Author(s):

SHAKHLO N. YARBAEVA ◽

PADMANABHA R. VELUGOTI ◽

HARSHAVARDHAN THIPPAREDDI ◽

JULIE A. ALBRECHT

Keyword(s):

Organic Acid ◽

Clostridium Perfringens ◽

Spore Germination ◽

Sodium Citrate ◽

Ground Beef ◽

E Coli ◽

Potassium Lactate ◽

Before And After ◽

Organic Acid Salts ◽

Acid Salts

Clostridium perfringens spore destruction, aerobic plate counts (APCs), and counts of Enterobacteriaceae, coliforms, and Escherichia coli during baking of sambusa (a traditional Tajik food) were evaluated. Control of germination and outgrowth of C. perfringens spores in sambusa during cooling at room or refrigerated temperatures was evaluated using organic acid salts (buffered sodium citrate [Ional] and 1 and 2% potassium lactate, wt/wt). Sambusa were prepared with 40 g of either inoculated or noninoculated meat and baked for 45 min at 180°C. For evaluation of destruction of C. perfringens spores during heating and germination and outgrowth of spores during cooling, ground beef was inoculated and mixed with a three-strain cocktail of C. perfringens spores. Aerobic bacteria, Enterobacteriaceae, coliforms, and E. coli were enumerated in noninoculated sambusa before and after baking and after cooling at room or refrigeration temperatures. After baking, APCs and Enterobacteriaceae and coliform counts were reduced by 4.32, 2.55, and 1.96 log CFU/g, respectively. E. coli counts were below detectable levels in ground beef and sambusa samples. Enterobacteriaceae, coliform, and E. coli counts were below detectable levels (<0.04 log CFU/g) in sambusa after cooling by both methods. Total C. perfringens populations increased (4.67 log CFU/g) during cooling at room temperature, but minimal increases (0.31 log CFU/g) were observed during cooling under refrigeration. Incorporation of 2% (wt/wt) buffered sodium citrate controlled C. perfringens spore germination and outgrowth (0.25 log CFU/g), whereas incorporation of up to 2% (wt/wt) potassium lactate did not prevent C. perfringens spore germination and outgrowth. Incorporation of organic acid salts at appropriate concentrations can prevent germination and outgrowth of C. perfringens in improperly cooled sambusa.

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Characterization of Clostridium perfringens Spores That Lack SpoVA Proteins and Dipicolinic Acid

Journal of Bacteriology ◽

10.1128/jb.00325-08 ◽

2008 ◽

Vol 190 (13) ◽

pp. 4648-4659 ◽

Cited By ~ 61

Author(s):

Daniel Paredes-Sabja ◽

Barbara Setlow ◽

Peter Setlow ◽

Mahfuzur R. Sarker

Keyword(s):

Water Content ◽

Uv Radiation ◽

Clostridium Perfringens ◽

Spore Germination ◽

Gastrointestinal Disease ◽

Dipicolinic Acid ◽

High Heat ◽

Wild Type ◽

Active Growth ◽

Moist Heat

ABSTRACT Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca2+ and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca2+ and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca2+ and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 103- to 104-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective α/β-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.

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Control of Clostridium perfringens Spores by Green Tea Leaf Extracts during Cooling of Cooked Ground Beef, Chicken, and Pork†

Journal of Food Protection ◽

10.4315/0362-028x-70.6.1429 ◽

2007 ◽

Vol 70 (6) ◽

pp. 1429-1433 ◽

Cited By ~ 33

Author(s):

VIJAY K. JUNEJA ◽

M. L. BARI ◽

Y. INATSU ◽

S. KAWAMOTO ◽

MENDEL FRIEDMAN

Keyword(s):

Green Tea ◽

Clostridium Perfringens ◽

Spore Germination ◽

Ground Beef ◽

Leaf Extracts ◽

Inhibition Of Growth ◽

Dependent Inhibition ◽

Leaf Powder ◽

Tea Extract ◽

Tea Leaf

We investigated the inhibition of Clostridium perfringens spore germination and outgrowth by two green tea extracts with low (green tea leaf powder [GTL]; 141 mg of total catechins per g of green tea extract) and high (green tea leaf extract [GTE]; 697 mg of total catechins per g of extract) catechin levels during abusive chilling of retail cooked ground beef, chicken, and pork. Green tea extracts were mixed into the thawed beef, chicken, and pork at concentrations of 0.5, 1.0, and 2.0% (wt/wt), along with a heat-activated (75°C for 20 min) three-strain spore cocktail to obtain a final concentration of ∼3 log spores per g. Samples (5 g) of the ground beef, chicken, and pork were then vacuum packaged and cooked to 71°Cfor1hina temperature-controlled water bath. Thereafter, the products were cooled from 54.4 to 7.2°C in 12, 15, 18, or 21 h, resulting in significant increases (P < 0.05) in the germination and outgrowth of C. perfringens populations in the ground beef, chicken, and pork control samples without GTL or GTE. Supplementation with 0.5 to 2% levels of GTL did not inhibit C. perfringens growth from spores. In contrast, the addition of 0.5 to 2% levels of GTE to beef, chicken, and pork resulted in a concentration-and time-dependent inhibition of C. perfringens growth from spores. At a 2% level of GTE, a significant (P < 0.05) inhibition of growth occurred at all chill rates for cooked ground beef, chicken, and pork. These results suggest that widely consumed catechins from green tea can reduce the potential risk of C. perfringens spore germination and outgrowth during abusive cooling from 54.4 to 7.2°C in 12, 15, 18, or 21 h of cooling for ground beef, chicken, and pork.

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Predictive Model for Clostridium perfringens Growth in Roast Beef during Cooling and Inhibition of Spore Germination and Outgrowth by Organic Acid Salts†‡

Journal of Food Protection ◽

10.4315/0362-028x-68.12.2594 ◽

2005 ◽

Vol 68 (12) ◽

pp. 2594-2605 ◽

Cited By ~ 19

Author(s):

MARCOS X. SÁNCHEZ-PLATA ◽

ALEJANDRO AMÉZQUITA ◽

ERIN BLANKENSHIP ◽

DENNIS E. BURSON ◽

VIJAY JUNEJA ◽

...

Keyword(s):

Organic Acid ◽

Predictive Model ◽

Clostridium Perfringens ◽

Spore Germination ◽

Sodium Citrate ◽

Sodium Lactate ◽

Processed Meat ◽

Roast Beef ◽

Organic Acid Salts ◽

Acid Salts

Spores of foodborne pathogens can survive traditional thermal processing schedules used in the manufacturing of processed meat products. Heat-activated spores can germinate and grow to hazardous levels when these products are improperly chilled. Germination and outgrowth of Clostridium perfringens spores in roast beef during chilling was studied following simulated cooling schedules normally used in the processed-meat industry. Inhibitory effects of organic acid salts on germination and outgrowth of C. perfringens spores during chilling and the survival of vegetative cells and spores under abusive refrigerated storage was also evaluated. Beef top rounds were formulated to contain a marinade (finished product concentrations: 1% salt, 0.2% potassium tetrapyrophosphate, and 0.2% starch) and then ground and mixed with antimicrobials (sodium lactate and sodium lactate plus 2.5% sodium diacetate and buffered sodium citrate and buffered sodium citrate plus 1.3% sodium diacetate). The ground product was inoculated with a three-strain cocktail of C. perfringens spores (NCTC 8238, NCTC 8239, and ATCC 10388), mixed, vacuum packaged, heat shocked for 20 min at 75°C, and chilled exponentially from 54.5 to 7.2°C in 9, 12, 15, 18, or 21 h. C. perfringens populations (total and spore) were enumerated after heat shock, during chilling, and during storage for up to 60 days at 10°C using tryptose-sulfite-cycloserine agar. C. perfringens spores were able to germinate and grow in roast beef (control, without any antimicrobials) from an initial population of ca. 3.1 log CFU/g by 2.00, 3.44, 4.04, 4.86, and 5.72 log CFU/g after 9, 12, 15, 18, and 21 h of exponential chilling. A predictive model was developed to describe sigmoidal C. perfringens growth curves during cooling of roast beef from 54.5 to 7.2°C within 9, 12, 15, 18, and 21 h. Addition of antimicrobials prevented germination and outgrowth of C. perfringens regardless of the chill times. C. perfringens spores could be recovered from samples containing organic acid salts that were stored up to 60 days at 10°C. Extension of chilling time to ≥9 h resulted in >1 log CFU/g growth of C. perfringens under anaerobic conditions in roast beef. Organic acid salts inhibited outgrowth of C. perfringens spores during chilling of roast beef when extended chill rates were followed. Although C. perfringens spore germination is inhibited by the antimicrobials, this inhibition may represent a hazard when such products are incorporated into new products, such as soups and chili, that do not contain these antimicrobials, thus allowing spore germination and outgrowth under conditions of temperature abuse.

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GerO, a Putative Na+/H+-K+ Antiporter, Is Essential for Normal Germination of Spores of the Pathogenic Bacterium Clostridium perfringens

Journal of Bacteriology ◽

10.1128/jb.00158-09 ◽

2009 ◽

Vol 191 (12) ◽

pp. 3822-3831 ◽

Cited By ~ 18

Author(s):

Daniel Paredes-Sabja ◽

Peter Setlow ◽

Mahfuzur R. Sarker

Keyword(s):

Clostridium Perfringens ◽

Spore Germination ◽

Dipicolinic Acid ◽

Ectopic Expression ◽

Monovalent Cation ◽

Wild Type ◽

Cell Compartment ◽

E Coli ◽

Modeled Structure ◽

Cation Transporters

ABSTRACT The genome of the pathogen Clostridium perfringens encodes two proteins, GerO and GerQ, homologous to monovalent cation transporters suggested to have roles in the germination of spores of some Bacillus species. GerO and GerQ were able to transport monovalent cations (K+ and/or Na+) in Escherichia coli, and gerO and gerQ were expressed only in the mother cell compartment during C. perfringens sporulation. C. perfringens spores lacking GerO were defective in germination with a rich medium, KCl, l-asparagine, and a 1:1 chelate of Ca2+ and dipicolinic acid (DPA), but not with dodecylamine, and the defect was prior to DPA release in germination. All defects in gerO spores were complemented by ectopic expression of wild-type gerO. Loss of GerQ had much smaller effects on spore germination, and these effects were most evident in spores also lacking GerO. A modeled structure of GerO was similar to that of the E. coli Na+/H+ antiporter NhaA, and GerO, but not GerQ contained two adjacent Asp residues thought to be important in the function of this group of cation transporters. Replacement of these adjacent Asp residues in GerO with Asn reduced the protein's ability to complement the germination defect in gerO spores but not the ability to restore cation transport to E. coli cells defective in K+ uptake. Together, these data suggest that monovalent cation transporters play some role in C. perfringens spore germination. However, it is not clear whether this role is directly in germination or perhaps in spore formation.

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Changes in the hydrophobic characteristics of Clostridium perfringens spores and spore coats by heat

Canadian Journal of Microbiology ◽

10.1139/m87-132 ◽

1987 ◽

Vol 33 (9) ◽

pp. 773-776 ◽

Cited By ~ 12

Author(s):

S. E. Craven ◽

L. C. Blankenship

Keyword(s):

Sodium Dodecyl Sulfate ◽

Clostridium Perfringens ◽

Spore Germination ◽

Sodium Dodecyl ◽

Spore Coat ◽

Vegetative Cells ◽

Partition System ◽

Dodecyl Sulfate ◽

Heat Activation

The hydrophobic characteristics of Clostridium perfringens NCTC 8679 spores were demonstrated by adherence to toluene in a toluene–aqueous partition system. Spores and spore coat preparations were hydrophobic. Vegetative cells and spores extracted with a dithiothreitol – sodium dodecyl sulfate treatment known to remove spore coats were not hydrophobic. A heat activation treatment (75 °C for 20 min) which promotes more rapid spore germination increased the hydrophobicity of intact spores and decreased that of isolated spore coats. The hydrophobic changes were reversed by washing and stabilized by 0.5% glutaraldehyde. Heat-induced hydrophobic changes were observed in spore coats prepared from spores that were preheated and washed before rupturing in a buffer containing glutaraldehyde. These results suggest the occurrence of a heat-induced change in the spore coat (possibly in the conformation of a macromolecule) which was stable only within the architectural confines of the intact spore.

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Control of Clostridium perfringens in Cooked Ground Beef by Carvacrol, Cinnamaldehyde, Thymol, or Oregano Oil during Chilling†

Journal of Food Protection ◽

10.4315/0362-028x-69.7.1546 ◽

2006 ◽

Vol 69 (7) ◽

pp. 1546-1551 ◽

Cited By ~ 44

Author(s):

VIJAY K. JUNEJA ◽

H. THIPPAREDDI ◽

MENDEL FRIEDMAN

Keyword(s):

Clostridium Perfringens ◽

Potential Risk ◽

Spore Germination ◽

Meat Product ◽

Ground Beef ◽

Grocery Store ◽

Water Bath ◽

The Other ◽

Oregano Oil ◽

Inhibit Spore Germination

Inhibition of Clostridium perfringens spore germination and outgrowth by carvacrol, cinnamaldehyde, thymol, and oregano oil was evaluated during abusive chilling of cooked ground beef (75% lean) obtained from a local grocery store. Test substances were mixed into thawed ground beef at concentrations of 0.1, 0.5, 1.0, or 2.0% (wt/wt) along with a heat-activated three-strain C. perfringens spore cocktail to obtain final spore concentrations of ca. 2.8 log spores per g. Aliquots (5 g) of the ground beef mixtures were vacuum-packaged and then cooked in a water bath, the temperature of which was raised to 60°C in 1 h. The products were cooled from 54.4 to 7.2°C in 12, 15, 18, or 21 h, resulting in 3.18, 4.64, 4.76, and 5.04 log CFU/g increases, respectively, in C. perfringens populations. Incorporation of test compounds (≥0.1%) into the beef completely inhibited C. perfringens spore germination and outgrowth (P ≤ 0.05) during exponential cooling of the cooked beef in 12 h. Longer chilling times (15, 18, and 21 h) required greater concentrations to inhibit spore germination and outgrowth. Cinnamaldehyde was significantly (P < 0.05) more effective (<1.0 log CFU/g growth) at a lower concentration (0.5%) at the most abusive chilling rate evaluated (21 h) than the other compounds. Incorporation of lower levels of these test compounds with other antimicrobials used in meat product formulations may reduce the potential risk of C. perfringens germination and outgrowth during abusive cooling regimes.

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Physiological role of carbon dioxide in spore germination of Clostridium perfringens S40

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2009.06.011 ◽

2009 ◽

Vol 108 (6) ◽

pp. 477-483 ◽

Cited By ~ 2

Author(s):

Shiro Kato ◽

Atsushi Masayama ◽

Tohru Yoshimura ◽

Hisashi Hemmi ◽

Hidenori Tsunoda ◽

...

Keyword(s):

Carbon Dioxide ◽

Clostridium Perfringens ◽

Spore Germination ◽

Physiological Role

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Inhibition of Clostridium perfringens Spore Germination and Outgrowth by Buffered Vinegar and Lemon Juice Concentrate during Chilling of Ground Turkey Roast Containing Minimal Ingredients†

Journal of Food Protection ◽

10.4315/0362-028x-73.3.470 ◽

2010 ◽

Vol 73 (3) ◽

pp. 470-476 ◽

Cited By ~ 20

Author(s):

CAROL VALENZUELA-MARTINEZ ◽

AIDA PENA-RAMOS ◽

VIJAY K. JUNEJA ◽

NAGESWARA RAO KORASAPATI ◽

DENNIS E. BURSON ◽

...

Keyword(s):

Clostridium Perfringens ◽

Spore Germination ◽

Lemon Juice ◽

Sea Salt ◽

Juice Concentrate ◽

Control Samples

Inhibition of Clostridium perfringens spore germination and outgrowth in ground turkey roast containing minimal ingredients (salt and sugar), by buffered vinegar (MOstatin V) and a blend (buffered) of lemon juice concentrate and vinegar (MOstatin LV) was evaluated. Ground turkey roast was formulated to contain sea salt (1.5%), turbinado sugar (0.5%), and various concentrations of MOstatin V (0.75, 1.25, or 2.5%) or MOstatin LV (1.5, 2.5, or 3.5%), along with a control (without MOstatins). The product was inoculated with a three-strain spore cocktail of C. perfringens to obtain initial spore levels of ca. 2.0 to 0.5 log CFU/g. Inoculated products were vacuum packaged, heat shocked for 20 min at 75°C, and cooled exponentially from 54.4 to 4.0°C in 6.5, 9, 12, 15, 18, or 21 h. In control samples without MOstatin V or MOstatin LV, C. perfringens populations reached 2.98, 4.50, 5.78, 7.05, 7.88, and 8.19 log CFU/g (corresponding increases of 0.51, 2.29, 3.51, 4.79, 5.55, and 5.93 log CFU/g) in 6.5, 9, 12, 15, 18, and 21 h of chilling, respectively. MOstatin V (2.5%) and MOstatin LV (3.5%) were effective in inhibiting C. perfringens spore germination and outgrowth in ground turkey roast to <1.0 log CFU/g during abusive chilling of the product within 21 h. Buffered vinegar and a blend (buffered) of lemon juice concentrate and vinegar were effective in controlling germination and outgrowth of C. perfringens spores in turkey roast containing minimal ingredients.

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