Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye

Laflamme, Christian; Ho, Jim; Veillette, Marc; Latrémoille, Marie-Chantal de; Verreault, Daniel; Mériaux, Anne; Duchaine, Caroline

doi:10.1007/s00203-004-0750-9

Cited by 41 publications

(35 citation statements)

References 33 publications

(27 reference statements)

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“…2). This taxonomic bias introduced during the Nycodenz cell extraction process has been noted previously (31) and may be related, in part, to the differential removal of spore-forming bacteria during the Nycodenz extraction process (spores have a higher density than vegetative cells [32]) or due to the tendency of some taxa to be more closely bound to mineral soil particles than other taxa (33).…”

Section: Resultsmentioning

confidence: 84%

Cell Size Distributions of Soil Bacterial and Archaeal Taxa

Guisado

Leff

Lauber

et al. 2013

Appl Environ Microbiol

104

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Cell size is a key ecological trait of soil microorganisms that determines a wide range of life history attributes, including the efficiency of nutrient acquisition. However, because of the methodological issues associated with determining cell sizes in situ, we have a limited understanding of how cell abundances vary across cell size fractions and whether certain microbial taxa have consistently smaller cells than other taxa. In this study, we extracted cells from three distinct soils and fractionated them into seven size ranges (5 m to 0.2 m) by filtration. Cell abundances in each size fraction were determined by direct microscopy, with the taxonomic composition of each size fraction determined by high-throughput sequencing of the 16S rRNA gene. Most of the cells were smaller than cells typically grown in culture, with 59 to 67% of cells <1.2 m in diameter. Furthermore, each size fraction harbored distinct bacterial and archaeal communities in each of the three soils, and many of the taxa exhibited distinct size distribution patterns, with the smaller size fractions having higher relative abundances of taxa that are rare or poorly characterized (including Acidobacteria, Gemmatimonadetes, Crenarchaeota, Verrucomicrobia, and Elusimicrobia). In general, there was a direct relationship between average cell size and culturability, with those soil taxa that are poorly represented in culture collections tending to be smaller. Size fractionation not only provides important insight into the life history strategies of soil microbial taxa but also is a useful tool to enable more focused investigations into those taxa that remain poorly characterized.

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Section: Resultsmentioning

confidence: 84%

Cell Size Distributions of Soil Bacterial and Archaeal Taxa

Guisado

Leff

Lauber

et al. 2013

Appl Environ Microbiol

104

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“…One additional outcome of the identification of these stable and severely coat-defective cotE gerE B. subtilis spores is that such spores provided an excellent reagent for determining the reason that spores often give faint peripheral staining with stains that are thought to be relatively specific for nucleic acids or membranes (15,20,34), as this could well be due to adsorption of these stains to coat protein. In addition, the relative lack of coat proteins from cotE gerE spores may allow the identification of the spore components responsible for the spore's significant autofluorescence, a property that has been suggested may be useful for spore detection (16,17).…”

Section: Discussionmentioning

confidence: 99%

Characterization of Spores of Bacillus subtilis That Lack Most Coat Layers

Ghosh¹,

Setlow²,

Wahome³

et al. 2008

J Bacteriol

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Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca 2؉ and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.

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“…In the presence of germinant, B. anthracis demonstrated significantly stronger staining with the membrane potential-sensitive dye DiOC 2 (29) than in the absence of germinant, indicating the establishment of a membrane potential by 30 min subsequent to germination initiation (Fig. 5C).…”

Section: Fig 3 the Inhibitory Action Of Nisin Is Irreversiblementioning

confidence: 94%

“…The B. anthracis spores were incubated as described under "Culture of B. anthracis spores," except for the presence of a fluorescent membrane potential-sensitive dye, 3-3Јdiethyloxacarbocyanine iodide (DiOC 2 ; 300 nM; Invitrogen, Carlsbad, CA) (29). At the indicated times, the membrane potential was assessed by measuring the increase in B. anthracis-associated DiOC 2 fluorescence by flow cytometry (EPICS XL-MCL flow cytometer; Beckman Coulter, Fullerton, CA), with excitation at 488 nm with an argon laser and measurement of the fluorescence emission through a band-pass filter at 525/20 nm.…”

mentioning

confidence: 99%

Inhibition of Bacillus anthracis Spore Outgrowth by Nisin

Gut

Prouty

Ballard

et al. 2008

Antimicrob Agents Chemother

100

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The lantibiotic nisin has previously been reported to inhibit the outgrowth of spores from several Bacillus species. However, the mode of action of nisin responsible for outgrowth inhibition is poorly understood. By using B. anthracis Sterne 7702 as a model, nisin acted against spores with a 50% inhibitory concentration (IC 50 ) and an IC 90 of 0.57 M and 0.90 M, respectively. Viable B. anthracis organisms were not recoverable from cultures containing concentrations of nisin greater than the IC 90 . These studies demonstrated that spores lose heat resistance and become hydrated in the presence of nisin, thereby ruling out a possible mechanism of inhibition in which nisin acts to block germination initiation. Rather, germination initiation is requisite for the action of nisin. This study also revealed that nisin rapidly and irreversibly inhibits growth by preventing the establishment of oxidative metabolism and the membrane potential in germinating spores. On the other hand, nisin had no detectable effects on the typical changes associated with the dissolution of the outer spore structures (e.g., the spore coats, cortex, and exosporium). Thus, the action of nisin results in the uncoupling of two critical sequences of events necessary for the outgrowth of spores: the establishment of metabolism and the shedding of the external spore structures.Lantibiotics are methyllanthionine-containing cationic antimicrobial peptides produced by several gram-positive bacteria (11). Nisin is a 34-amino-acid peptide produced by Lactococcus lactis subsp. lactis (ATCC 11454), which has emerged as an important prototype for the study of the novel antibacterial properties and structure-activity relationships characteristic of the lantibiotics (5, 33). Like all lantibiotics, nisin is ribosomally translated and is then posttranslationally modified to generate three noncyclic nonproteogenic amino acids, dehydroalanine, and dehydrobutyrine and five lanthionine or methyllanthionine thioether rings (11).The utility of nisin derives from its capacity to act upon gram-positive bacteria by two entirely different mechanisms (15,46). Nisin forms pores in lipid membranes (46), but it also functions as a transglycosylase inhibitor that disrupts cell wall biosynthesis via lipid II binding and mislocalization (21,55). Because it functions as a "two-edged sword," microbes have been relatively refractory to the emergence of resistance to nisin, despite its widespread and persistent use as a preservative in the food industry (15,46).An additional and poorly understood activity of nisin is its capacity to prevent the outgrowth of spores from several grampositive bacteria, including several Bacillus species (9, 10, 40, 42). To date, nisin inhibition of Bacillus spore outgrowth has been documented by various methods, including the spectrophotometric measurement of liquid culture turbidity (3), the enumeration of CFU (4,14,32,35,43), well diffusion assays on solid agar (14, 39), and microscopic observations (41). Although these approaches are useful, they...

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Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye

Cited by 41 publications

References 33 publications

Cell Size Distributions of Soil Bacterial and Archaeal Taxa

Cell Size Distributions of Soil Bacterial and Archaeal Taxa

Characterization of Spores of Bacillus subtilis That Lack Most Coat Layers

Inhibition of Bacillus anthracis Spore Outgrowth by Nisin

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