Role of GerKB in Germination and Outgrowth of Clostridium perfringens Spores

ABSTRACT Previous work indicated that Clostridium perfringens gerKA gerKC spores germinate significantly, suggesting that gerKB also has a role in C. perfringens spore germination. We now find that (i) gerKB was expressed only during sporulation, likely in the forespore; (ii) gerKB spores germinated like wild-type spores with nonnutrient germinants and with high concentrations of nutrients but more slowly with low nutrient concentrations; and (iii) gerKB spores had lower colony-forming efficiency and slower outgrowth than wild-type spores. These results suggest that GerKB plays an auxiliary role in spore germination under some conditions and is required for normal spore viability and outgrowth.

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SleC Is Essential for Cortex Peptidoglycan Hydrolysis during Germination of Spores of the Pathogenic Bacterium Clostridium perfringens

Journal of Bacteriology ◽

10.1128/jb.01832-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2711-2720 ◽

Cited By ~ 65

Author(s):

Daniel Paredes-Sabja ◽

Peter Setlow ◽

Mahfuzur R. Sarker

Keyword(s):

Clostridium Perfringens ◽

Spore Germination ◽

Dipicolinic Acid ◽

Pathogenic Bacterium ◽

Wild Type ◽

Lytic Enzymes ◽

Colony Forming Efficiency ◽

Forming Efficiency ◽

Peptidoglycan Hydrolysis

ABSTRACT Clostridial spore germination requires degradation of the spore's peptidoglycan (PG) cortex by cortex-lytic enzymes (CLEs), and two Clostridium perfringens CLEs, SleC and SleM, degrade cortex PG in vitro. We now find that only SleC is essential for cortex hydrolysis and viability of C. perfringens spores. C. perfringens sleC spores did not germinate completely with nutrients, KCl, or a 1:1 chelate of Ca2+ and dipicolinic acid (Ca-DPA), and the colony-forming efficiency of sleC spores was 103-fold lower than that of wild-type spores. However, sleC spores incubated with various germinants released most of their DPA, although slower than wild-type or sleM spores, and DPA release from sleC sleM spores was very slow. In contrast, germination and viability of sleM spores were similar to that of wild-type spores, although sleC sleM spores had 105-fold-lower viability. These results allow the following conclusions about C. perfringens spore germination: (i) SleC is essential for cortex hydrolysis; (ii) although SleM can degrade cortex PG in vitro, this enzyme is not essential; (iii) action of SleC alone or with SleM can accelerate DPA release; and (iv) Ca-DPA does not trigger spore germination by activation of CLEs.

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Clostridium perfringens Spore Germination: Characterization of Germinants and Their Receptors

Journal of Bacteriology ◽

10.1128/jb.01748-07 ◽

2007 ◽

Vol 190 (4) ◽

pp. 1190-1201 ◽

Cited By ~ 105

Author(s):

Daniel Paredes-Sabja ◽

J. Antonio Torres ◽

Peter Setlow ◽

Mahfuzur R. Sarker

Keyword(s):

Clostridium Perfringens ◽

Spore Germination ◽

Dipicolinic Acid ◽

Food Poisoning ◽

Wild Type ◽

Active Growth ◽

Food Borne ◽

High Concentrations ◽

Auxiliary Role

ABSTRACT Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of l-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, l-asparagine triggered spore germination in C-cpe isolates only; and (ii) l-alanine or l-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with l-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.

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Saccharomyces cerevisiae Checkpoint Genes MEC1, RAD17 and RAD24 Are Required for Normal Meiotic Recombination Partner Choice

Genetics ◽

10.1093/genetics/153.2.607 ◽

1999 ◽

Vol 153 (2) ◽

pp. 607-620 ◽

Cited By ~ 4

Author(s):

Jeremy M Grushcow ◽

Teresa M Holzen ◽

Ken J Park ◽

Ted Weinert ◽

Michael Lichten ◽

...

Keyword(s):

Meiotic Recombination ◽

Mitotic Checkpoint ◽

Partner Choice ◽

Wild Type ◽

Spore Viability ◽

Checkpoint Signaling ◽

Ectopic Recombination ◽

Meiotic Progression ◽

Checkpoint Genes

Abstract Checkpoint gene function prevents meiotic progression when recombination is blocked by mutations in the recA homologue DMC1. Bypass of dmc1 arrest by mutation of the DNA damage checkpoint genes MEC1, RAD17, or RAD24 results in a dramatic loss of spore viability, suggesting that these genes play an important role in monitoring the progression of recombination. We show here that the role of mitotic checkpoint genes in meiosis is not limited to maintaining arrest in abnormal meioses; mec1-1, rad24, and rad17 single mutants have additional meiotic defects. All three mutants display Zip1 polycomplexes in two- to threefold more nuclei than observed in wild-type controls, suggesting that synapsis may be aberrant. Additionally, all three mutants exhibit elevated levels of ectopic recombination in a novel physical assay. rad17 mutants also alter the fraction of recombination events that are accompanied by an exchange of flanking markers. Crossovers are associated with up to 90% of recombination events for one pair of alleles in rad17, as compared with 65% in wild type. Meiotic progression is not required to allow ectopic recombination in rad17 mutants, as it still occurs at elevated levels in ndt80 mutants that arrest in prophase regardless of checkpoint signaling. These observations support the suggestion that MEC1, RAD17, and RAD24, in addition to their proposed monitoring function, act to promote normal meiotic recombination.

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Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

Journal of Bacteriology ◽

10.1128/jb.01613-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1565-1572 ◽

Cited By ~ 82

Author(s):

Venkata Ramana Vepachedu ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Small Molecules ◽

Spore Germination ◽

Dipicolinic Acid ◽

Temperature Sensitive ◽

Wild Type ◽

Ph Optimum ◽

Inner Membrane ◽

Temperature Sensitive Mutants

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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Effect of Small, Acid-Soluble Proteins on Spore Resistance and Germination under a Combination of Pressure and Heat Treatment

Journal of Food Protection ◽

10.4315/0362-028x-70.9.2168 ◽

2007 ◽

Vol 70 (9) ◽

pp. 2168-2171

Author(s):

JONG-KYUNG LEE ◽

SARA MOVAHEDI ◽

STEPHEN E. HARDING ◽

BERNARD M. MACKEY ◽

WILLIAM M. WAITES

Keyword(s):

Heat Treatment ◽

High Pressure ◽

High Temperature ◽

Spore Germination ◽

Soluble Proteins ◽

Wild Type ◽

Spore Resistance ◽

Pressure Resistance ◽

The One

To find the range of pressure required for effective high-pressure inactivation of bacterial spores and to investigate the role of α/β-type small, acid-soluble proteins (SASP) in spores under pressure treatment, mild heat was combined with pressure (room temperature to 65°C and 100 to 500 MPa) and applied to wild-type and SASP-α−/β− Bacillus subtilis spores. On the one hand, more than 4 log units of wild-type spores were reduced after pressurization at 100 to 500 MPa and 65°C. On the other hand, the number of surviving mutant spores decreased by 2 log units at 100 MPa and by more than 5 log units at 500 MPa. At 500 MPa and 65°C, both wild-type and mutant spore survivor counts were reduced by 5 log units. Interestingly, pressures of 100, 200, and 300 MPa at 65°C inactivated wild-type SASP-α+/β+ spores more than mutant SASP-α−/β− spores, and this was attributed to less pressure-induced germination in SASP-α−/β− spores than in wild-type SASP-α+/β+ spores. However, there was no difference in the pressure resistance between SASP-α+/β+ and SASP-α−/β− spores at 100 MPa and ambient temperature (approximately 22°C) for 30 min. A combination of high pressure and high temperature is very effective for inducing spore germination, and then inactivation of the germinated spore occurs because of the heat treatment. This study showed that α/β-type SASP play a role in spore inactivation by increasing spore germination under 100 to 300 MPa at high temperature.

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The Role of Nerve Growth Factor in Maintaining Proliferative Capacity, Colony-Forming Efficiency, and the Limbal Stem Cell Phenotype

Stem Cells ◽

10.1002/stem.2921 ◽

2018 ◽

Vol 37 (1) ◽

pp. 139-149 ◽

Cited By ~ 8

Author(s):

Sai Kolli ◽

Sanja Bojic ◽

Ali E. Ghareeb ◽

Marzena Kurzawa-Akanbi ◽

Francisco C. Figueiredo ◽

...

Keyword(s):

Stem Cell ◽

Nerve Growth Factor ◽

Growth Factor ◽

Cell Phenotype ◽

Proliferative Capacity ◽

Colony Forming Efficiency ◽

Forming Efficiency ◽

Limbal Stem Cell ◽

Stem Cell Phenotype

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Role of Chromosomal and Plasmid-Borne Receptor Homologues in the Response of Bacillus megaterium QM B1551 Spores to Germinants

Journal of Bacteriology ◽

10.1128/jb.00110-07 ◽

2007 ◽

Vol 189 (12) ◽

pp. 4375-4383 ◽

Cited By ~ 40

Author(s):

Graham Christie ◽

Christopher R. Lowe

Keyword(s):

Gene Cluster ◽

Bacillus Megaterium ◽

Spore Germination ◽

Deletion Mutation ◽

Inorganic Salts ◽

Chimeric Receptor ◽

Wild Type ◽

Germination Response ◽

Receptor Component

ABSTRACT Spores of Bacillus megaterium QM B1551 germinate in response to a number of trigger compounds, including glucose, proline, leucine, and inorganic salts. An approximate 6-kb region of the 165-kb plasmid was found to harbor a tricistronic receptor operon, gerU, and a monocistronic receptor component, gerVB. The gerU operon was observed to complement the germination response in plasmidless strain PV361 to glucose and leucine, with KBr acting as a cogerminant. Proline recognition is conferred by the monocistronic gerVB gene, the presence of which also improves the germination response to other single-trigger compounds. A chimeric receptor, GerU*, demonstrates interchangeability between receptor components and provides evidence that it is the B protein of the receptor that determines germinant specificity. Introduction of the gerU/gerVB gene cluster to B. megaterium KM extends the range of germinants recognized by this strain to include glucose, proline, and KBr in addition to alanine and leucine. A chromosomally encoded receptor, GerA, the B component of which is predicted to be truncated, was found to be functionally redundant. Similarly, the plasmid-borne antiporter gene, grmA, identified previously as being essential for germination in QM B1551, did not complement the germination defect in the plasmidless variant PV361. Wild-type spores carrying an insertion-deletion mutation in this cistron germinated normally; thus, the role of GrmA in spore germination needs to be reevaluated in this species.

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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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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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Characterization of Acp, a Peptidoglycan Hydrolase of Clostridium perfringens with N-Acetylglucosaminidase Activity That Is Implicated in Cell Separation and Stress-Induced Autolysis

Journal of Bacteriology ◽

10.1128/jb.01546-09 ◽

2010 ◽

Vol 192 (9) ◽

pp. 2373-2384 ◽

Cited By ~ 20

Author(s):

Emilie Camiade ◽

Johann Peltier ◽

Ingrid Bourgeois ◽

Evelyne Couture-Tosi ◽

Pascal Courtin ◽

...

Keyword(s):

Mutant Strain ◽

Clostridium Perfringens ◽

Vegetative Growth ◽

Cell Separation ◽

Catalytic Domain ◽

Peptidoglycan Hydrolase ◽

Wild Type ◽

Induced Autolysis

ABSTRACT This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N-acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.

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