A modified reagent for dipicolinic acid analysis

Rotman, Yaron; Fields, M. L.

doi:10.1016/0003-2697(68)90272-8

Cited by 91 publications

(79 citation statements)

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“…The DPA remaining in spores after treatment with ethanol, acid or alkali or after spore germination was assayed as described previously (Rotman and Fields 1967;Tennen et al 2000), as was the total hexosamine content of dormant and germinated spores and the amount of hexosamine-containing material released into the supernatant fraction upon spore germination; the latter value is a measure of spore cortex degradation (Popham et al 1996;Tennen et al 2000). Light production during germination of spores containing the LuxAB proteins was measured as described by Ciarciaglini et al (2000).…”

Section: Analytical Methods and Spore Germinationmentioning

confidence: 99%

Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

et al. 2002

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Aims: To determine the mechanisms of killing of Bacillus subtilis spores by ethanol or strong acid or alkali. Methods and Results: Killing of B. subtilis spores by ethanol or strong acid or alkali was not through DNA damage and the spore coats did not protect spores against these agents. Spores treated with ethanol or acid released their dipicolinic acid (DPA) in parallel with spore killing and the core wet density of ethanol-or acid-killed spores fell to a value close to that for untreated spores lacking DPA. The core regions of spores killed by these two agents were stained by nucleic acid stains that do not penetrate into the core of untreated spores and acid-killed spores appeared to have ruptured. Spores killed by these two agents also did not germinate in nutrient and nonnutrient germinants and were not recovered by lysozyme treatment. Spores killed by alkali did not lose their DPA, did not exhibit a decrease in their core wet density and their cores were not stained by nucleic acid stains. Alkali-killed spores released their DPA upon initiation of spore germination, but did not initiate metabolism and degraded their cortex very poorly. However, spores apparently killed by alkali were recovered by lysozyme treatment. Conclusions: The data suggest that spore killing by ethanol and strong acid involves the disruption of a spore permeability barrier, while spore killing by strong alkali is due to the inactivation of spore cortex lytic enzymes. Significance and Impact of the Study: The results provide further information on the mechanisms of spore killing by various chemicals.

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Section: Analytical Methods and Spore Germinationmentioning

confidence: 99%

Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

et al. 2002

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“…The spores produced by strains FB72, PS832, and PS3317 were harvested, purified, and stored as described previously (16,18), and all of the spore preparations used were free (Ն96%) of sporulating cells, germinated spores, and cell debris. The DPA contents of the purified spores were determined as described previously (16,18,22), and as found previously (18), ger3 spoVF spores lacked DPA, as did ger3 spoVA spores ( Table 1). The core wet densities of the various types of spores, which reflected the core water contents of the spores (the higher the spore core wet density, the lower the spore core water content) (7), were determined as described previously (12).…”

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confidence: 89%

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

et al. 2002

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Bacillus subtilis cells with mutations in the spoVA operon do not complete sporulation. However, a spoVA strain with mutations that remove all three of the spore's functional nutrient germinant receptors (termed the ger3 mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation. ger3 spoVA and sleB spoVA spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or ger3 spores. These properties of ger3 spoVA and sleB spoVA spores are identical to those of ger3 spoVF and sleB spoVF spores that lack DPA due to deletion of the spoVF operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in ger3 spoVF spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into ger3 spoVA spores. These data indicate that one or more products of the spoVA operon are involved in DPA transport into the developing forespore during sporulation.A characteristic feature of the endospores of various Bacillus and Clostridium species is the presence of high levels of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) (2). DPA generally comprises Ն10% of the dry weight of these spores, and most of the DPA is likely in a 1:1 chelate with divalent cations, predominantly Ca 2ϩ (2). DPA is synthesized in the mother cell compartment of a sporulating cell from an intermediate in the lysine biosynthetic pathway, and the final synthetic step is catalyzed by DPA synthetase, the product of the two cistrons of the spoVF operon (1, 2, 3). DPA is located in the spore protoplast or core and is excreted in the first minute of spore germination (2, 21).In strains lacking DPA (for example, spoVF strains of Bacillus subtilis) sporulation is not completed as the developing spores lyse during sporulation (2,3,4,18). However, spoVF spores can be stabilized by additional mutations that remove either the three functional nutrient germinant receptors (termed the ger3 mutations) or a major spore cortex lytic enzyme, SleB (17,18,19). ger3 spoVF and sleB spoVF spores have higher levels of core water and consequently are less wet heat resistant than wild-type dormant spores (7,17,18; B. Setlow and P. Setlow, unpublished data). This suggests that in addition to stabilizing the spore's dormant state, DPA is also important in reducing the spore's core water content and thereby increasing its heat resistance (7).Although the location and pathway of DPA synthesis are known and there has been some general indication of the function of DPA in the developing and dormant spore, it is unclear how DPA gets into the developing forespore from its site of synthesis in the mother cell. It is also unclear how the dormant spore's DPA is excreted in the first minute of spore germination. The latter process is of interest, since DPA movement out of the dormant spore is regulated. DPA is normally retained in the dormant spore for long periods, but it is excreted within minutes either when nutrients bind to germinant recept...

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“…Spores with or without PON treatment were tested for mutagenesis to asporogeny or auxotrophy as described by Fairhead et al (1993). The pyridine-2,6-dicarboxylic acid [dipicolinic acid (DPA)] content of spores with or without PON treatment was assayed after DPA extraction by boiling as described (Rotman & Fields, 1967 ;Nicholson & Setlow, 1990). Spores with or without prior PON or Sterilox treatment were incubated in water at an OD '!!…”

Section: Bacterial Strains and Isolation Of Spores And Growing Cellsmentioning

confidence: 99%

Killing of spores of Bacillus subtilis by peroxynitrite appears to be caused by membrane damage

et al. 2002

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During an infection of a higher eukaryote, dormant spores of a Bacillus species have been previously shown to be present in cells that can generate the toxic agent peroxynitrite (PON). Dormant spores of Bacillus subtilis were much more resistant to killing by PON than were growing cells, and spore-coat alteration or removal greatly decreased PON resistance. Spores were not killed by PON through DNA damage and lost no dipicolinic acid (DPA) during PON treatment. However, PON-killed spores lost DPA during subsequent heat treatments that caused much less DPA release from untreated spores. Although dead, the PONkilled spores germinated and initiated metabolism but never went through outgrowth ; the great majority of germinated PON-killed spores also took up propidium iodide, indicating that they had suffered significant membrane damage and were dead. Together these data suggest that spore killing by PON is through some type of damage to the spore's inner membrane.

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A modified reagent for dipicolinic acid analysis

Cited by 91 publications

References 1 publication

Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

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

Killing of spores of Bacillus subtilis by peroxynitrite appears to be caused by membrane damage

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