scholarly journals Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

2010 ◽  
Vol 192 (14) ◽  
pp. 3608-3619 ◽  
Author(s):  
Pengfei Zhang ◽  
Will Garner ◽  
Xuan Yi ◽  
Ji Yu ◽  
Yong-qing Li ◽  
...  
Keyword(s):  
Dipicolinic Acid ◽  
Great Majority ◽  
Bacillus Species ◽  
Published Data ◽  
Laser Tweezers ◽  
Wild Type ◽  
Factors Affecting ◽  
Number Of Factors ◽  
Heat Activation ◽  
Acid Release

ABSTRACT The simultaneous nutrient germination of hundreds of individual wild-type spores of three Bacillus species and a number of Bacillus subtilis strains has been measured by two new methods, and rates of release of the great majority of the large pool of dipicolinic acid (DPA) from individual spores of B. subtilis strains has been measured by Raman spectroscopy with laser tweezers. The results from these analyses and published data have allowed a number of significant conclusions about the germination of spores of Bacillus species as follows. (i) The time needed for release of the great majority of a Bacillus spore's DPA once rapid DPA release had begun (ΔT release) during nutrient germination was independent of the concentration of nutrient germinant used, the level of the germinant receptors (GRs) that recognize nutrient germinants used and heat activation prior to germination. Values for ΔT release were generally 0.5 to 3 min at 25 to 37°C for individual wild-type spores. (ii) Despite the conclusion above, germination of individual spores in populations was very heterogeneous, with some spores in wild-type populations completing germination ≥15-fold slower than others. (iii) The major factor in the heterogeneity in germination of individual spores in populations was the highly variable lag time, T lag, between mixing spores with nutrient germinants and the beginning of ΔT release. (iv) A number of factors decrease spores' T lag values including heat activation, increased levels of GRs/spore, and higher levels of nutrient germinants. These latter factors appear to affect the level of activated GRs/spore during nutrient germination. (v) The conclusions above lead to the simple prediction that a major factor causing heterogeneity in Bacillus spore germination is the number of functional GRs in individual spores, a number that presumably varies significantly between spores in populations.

scholarly journals Studies of the Commitment Step in the Germination of Spores of Bacillus Species

2010 ◽  
Vol 192 (13) ◽  
pp. 3424-3433 ◽  
Author(s):  
Xuan Yi ◽  
Peter Setlow
Keyword(s):  
Spore Germination ◽  
Dipicolinic Acid ◽  
Lag Time ◽  
Bacillus Species ◽  
Lytic Enzymes ◽  
Large Heterogeneity ◽  
Heat Activation ◽  
Lag Times ◽  
The Times ◽  
Insight Into

ABSTRACT Spores of Bacillus species are said to be committed when they continue through nutrient germination even when germinants are removed or their binding to spores' nutrient germinant receptors (GRs) is both reversed and inhibited. Measurement of commitment and the subsequent release of dipicolinic acid (DPA) during nutrient germination of spores of Bacillus cereus and Bacillus subtilis showed that heat activation, increased nutrient germinant concentrations, and higher average levels of GRs/spore significantly decreased the times needed for commitment, as well as lag times between commitment and DPA release. These lag times were also decreased dramatically by the action of one of the spores' two redundant cortex lytic enzymes (CLEs), CwlJ, but not by the other CLE, SleB, and CwlJ action did not affect the timing of commitment. The timing of commitment and the lag time between commitment and DPA release were also dependent on the specific GR activated to cause spore germination. For spore populations, the lag times between commitment and DPA release were increased significantly in spores that germinated late compared to those that germinated early, and individual spores that germinated late may have had lower appropriate GR levels/spore than spores that germinated early. These findings together provide new insight into the commitment step in spore germination and suggest several factors that may contribute to the large heterogeneity among the timings of various events in the germination of individual spores in spore populations.

scholarly journals Levels of Glycine Betaine in Growing Cells and Spores of Bacillus Species and Lack of Effect of Glycine Betaine on Dormant Spore Resistance

2006 ◽  
Vol 188 (8) ◽  
pp. 3153-3158 ◽  
Author(s):  
Charles A. Loshon ◽  
Paul G. Wahome ◽  
Mark W. Maciejewski ◽  
Peter Setlow
Keyword(s):  
Glycine Betaine ◽  
Dipicolinic Acid ◽  
High Salt ◽  
Bacillus Species ◽  
Low Molecular Weight ◽  
Wild Type ◽  
Spore Resistance ◽  
Dormant Spore ◽  
Low Levels ◽  
Very High

ABSTRACT Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased ∼7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and ≥20-fold lower in B. cereus and B. megaterium spores. Athough GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.

scholarly journals A Polysaccharide Deacetylase Gene (pdaA) Is Required for Germination and for Production of Muramic δ-Lactam Residues in the Spore Cortex of Bacillus subtilis

2002 ◽  
Vol 184 (21) ◽  
pp. 6007-6015 ◽  
Author(s):  
Tatsuya Fukushima ◽  
Hiroki Yamamoto ◽  
Abdelmadjid Atrih ◽  
Simon J. Foster ◽  
Junichi Sekiguchi
Keyword(s):  
Bacillus Subtilis ◽  
Phase Contrast ◽  
Biosynthetic Pathway ◽  
Wild Type Strain ◽  
Dipicolinic Acid ◽  
Northern Hybridization ◽  
Predicted Amino Acid Sequence ◽  
Wild Type ◽  
Contrast Microscopy ◽  
Acid Release

ABSTRACT The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases. β-Galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by EσG RNA polymerase. pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of l-alanine. Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed. In particular, the release of hexosamine-containing materials was lacking in the pdaA mutant. Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic δ-lactam. A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B. subtilis spores. The biosynthetic pathway of muramic δ-lactam is discussed.

scholarly journals The Bacillus anthracis SleL (YaaH) Protein Is an N-Acetylglucosaminidase Involved in Spore Cortex Depolymerization

2008 ◽  
Vol 190 (23) ◽  
pp. 7601-7607 ◽  
Author(s):  
Emily A. Lambert ◽  
David L. Popham
Keyword(s):  
Bacillus Anthracis ◽  
Dipicolinic Acid ◽  
Diaminopimelic Acid ◽  
Lytic Enzyme ◽  
Wild Type ◽  
Lytic Enzymes ◽  
Spore Viability ◽  
Chromatography Analysis ◽  
Protective Layers ◽  
Acid Release

ABSTRACT Bacillus anthracis spores, the infectious agents of anthrax, are notoriously difficult to remove from contaminated areas because they are resistant to many eradication methods. These resistance properties are due to the spore's dehydration and dormancy and to the multiple protective layers surrounding the spore core, one of which is the cortex. In order for B. anthracis spores to germinate and resume growth, the cortex peptidoglycan must be depolymerized. This study reports on analyses of sleL (yaaH), which encodes a cortex-lytic enzyme. The inactivation of sleL does not affect vegetative growth, spore viability, or the initial stages of germination, including dipicolinic acid release. However, mutant spores exhibit a slight delay in the loss of optical density compared to that of wild-type spores. Mutants also retain more diaminopimelic acid and N-acetylmuramic acid during germination than wild-type spores, suggesting that the cortex peptidoglycan is not being hydrolyzed as rapidly. This finding is supported by high-pressure liquid chromatography analysis of the peptidoglycan structure used to confirm that SleL acts as an N-acetylglucosaminidase. When sleL is inactivated, the cortex peptidoglycan is not depolymerized into small muropeptides but instead is retained within the spore as large fragments. In the absence of the sleL-encoded N-acetylglucosaminidase, other cortex-lytic enzymes break down the cortex peptidoglycan sufficiently to allow rapid germination and outgrowth.

scholarly journals Superdormant Spores of Bacillus Species Have Elevated Wet-Heat Resistance and Temperature Requirements for Heat Activation

2009 ◽  
Vol 191 (18) ◽  
pp. 5584-5591 ◽  
Author(s):  
Sonali Ghosh ◽  
Pengfei Zhang ◽  
Yong-qing Li ◽  
Peter Setlow
Keyword(s):  
Heat Resistance ◽  
Dipicolinic Acid ◽  
Bacillus Species ◽  
Activation Temperature ◽  
New Information ◽  
Temperature Requirements ◽  
Wet Heat ◽  
Dormant Spore ◽  
Heat Activation ◽  
Temperature Optima

ABSTRACT Purified superdormant spores of Bacillus cereus, B. megaterium, and B. subtilis isolated after optimal heat activation of dormant spores and subsequent germination with inosine, d-glucose, or l-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15°C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations.

scholarly journals Isolation and Characterization of Superdormant Spores of Bacillus Species

2009 ◽  
Vol 191 (6) ◽  
pp. 1787-1797 ◽  
Author(s):  
Sonali Ghosh ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Bacillus Megaterium ◽  
Dipicolinic Acid ◽  
Nutrient Concentrations ◽  
Bacillus Species ◽  
Nutrient Levels ◽  
Isolation And Characterization ◽  
High Nutrient ◽  
Heat Activation

ABSTRACT Superdormant spores of Bacillus subtilis and Bacillus megaterium were isolated in 4 to 12% yields following germination with high nutrient levels that activated one or two germinant receptors. These superdormant spores did not germinate with the initial nutrients or those that stimulated other germinant receptors, and the superdormant spores' defect was not genetic. The superdormant spores did, however, germinate with Ca2+-dipicolinic acid or dodecylamine. Although these superdormant spores did not germinate with high levels of nutrients that activated one or two nutrient germinant receptors, they germinated with nutrient mixtures that activated more receptors, and using high levels of nutrient mixtures activating more germinant receptors decreased superdormant spore yields. The use of moderate nutrient levels to isolate superdormant spores increased their yields; the resultant spores germinated poorly with the initial moderate nutrient concentrations, but they germinated well with high nutrient concentrations. These findings suggest that the levels of superdormant spores in populations depend on the germination conditions used, with fewer superdormant spores isolated when better germination conditions are used. These findings further suggest that superdormant spores require an increased signal for triggering spore germination compared to most spores in populations. One factor determining whether a spore is superdormant is its level of germinant receptors, since spore populations with higher levels of germinant receptors yielded lower levels of superdormant spores. A second important factor may be heat activation of spore populations, since yields of superdormant spores from non-heat-activated spore populations were higher than those from optimally activated spores.

scholarly journals Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

2015 ◽  
Vol 197 (6) ◽  
pp. 1095-1103 ◽  
Author(s):  
Shiwei Wang ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
Spore Germination ◽  
Dipicolinic Acid ◽  
Lytic Enzyme ◽  
Early Event ◽  
Laser Tweezers ◽  
Wild Type ◽  
Content Type ◽  
Rapid Release ◽  
Contrast Microscopy ◽  
Bacillus Spores

When exposed to nutrient or nonnutrient germinants, individualBacillusspores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ∼20% of spore DPA) from individual spores that takes place prior to the lag time,Tlag, when spores begin rapid release of remaining DPA. Major conclusions from this work withBacillus subtilisspores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous timeT1; (ii) the period of slow DPA leakage (ΔTleakage=Tlag−T1) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreasedT1times but increased values of ΔTleakage; (iv) upon germination withl-valine for 10 min followed by addition ofd-alanine to block further germination, all germinated spores hadT1times of less than 10 min, suggesting thatT1is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreasedT1andTlagtimes but not the amount of DPA released in ΔTleakage; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ΔTleakagetimes in which more DPA was released; and (vii) there was slow DPA leakage early in germination ofB. subtilisspores by the nonnutrients CaDPA and dodecylamine and in nutrient germination ofBacillus cereusandBacillus megateriumspores. Overall, these findings have identified and characterized a new early event inBacillusspore germination.

Impact of climatic conditions on the parameters of failure flow of military vehicles

2021 ◽  
pp. 095440702110202
Author(s):  
Nikolaj Dobrzinskij ◽  
Algimantas Fedaravicius ◽  
Kestutis Pilkauskas ◽  
Egidijus Slizys
Keyword(s):  
Climatic Factors ◽  
Armed Forces ◽  
Climatic Conditions ◽  
Operating Conditions ◽  
Technical Equipment ◽  
Factors Affecting ◽  
Military Vehicles ◽  
Equipment Reliability ◽  
Number Of Factors ◽  
The Impact

Relevance of the article is based on participation of armed forces in various operations and exercises, where reliability of machinery is one of the most important factors. Transportation of soldiers as well as completion of variety of tasks is ensured by properly functioning technical equipment. Reliability of military vehicles – armoured SISU E13TP Finnish built and HMMWV M1025 USA built were selected as the object of the article. Impact of climatic conditions on reliability of the vehicles exploited in southwestern part of the Atlantic continental forest area is researched by a case study of the vehicles exploitation under conditions of the climate of Lithuania. Reliability of military vehicles depends on a number of factors such as properties of the vehicles and external conditions of their operation. Their systems and mechanisms are influenced by a number of factors that cause different failures. Climatic conditions represent one of the factors of operating load which is directly dependent on the climate zone. Therefore, assessment of the reliability is started with the analysis of climatic factors affecting operating conditions of the vehicles. Relationship between the impact of climatic factors and failure flow of the vehicles is presented and discussed.

Biodynamic Modeling of a Pedestrian Impact With a Rigid Frontal Guard of a Utility Vehicle

2010 ◽  
Author(s):  
Rasoul Moradi ◽  
Chandrashekhar K. Thorbole ◽  
Michael McCoy ◽  
Hamid M. Lankarani
Keyword(s):  
Front Surface ◽  
Body Region ◽  
Computational Technique ◽  
Surface Model ◽  
Published Data ◽  
Factors Affecting ◽  
Light Trucks ◽  
Accident Data ◽  
Facet Surface ◽  
The Impact

Accident data reveals that in most pedestrian accidents, the pedestrian head and lower extremity are vulnerable to serious injuries. The vehicle front geometry profile as well as the impact speed are important factors affecting the pedestrian kinematics and injury potential. In the US, accident data also shows that the fatality rate for pedestrian/light trucks and vans (LTV) impact is greater than that for the pedestrian/passenger-car impact. Addition of a front guard on light trucks and sports utility vehicles to mitigate damage during off-road activity or to provide mounting points for extra lights, makes the pedestrian more vulnerable to the impact. In this paper, a computational technique is utilized to study the influence of the added front guard on the impacted pedestrian. A CAD model of a typical commercial frontal guard is developed and converted into a rigid facet model, and attached to the vehicle front. The validated standing dummy model in the MADYMO code is used to simulate a pedestrian, and the rigid facet-surface model of a pickup truck is used to generate a vehicle front surface. This computational model is validated by comparing the pedestrian kinematics with the published data. This study demonstrates that the pedestrian mid body region is more vulnerable with the addition of guard on the vehicle. The result from this study facilitates a better understanding of a guard design and its geometry profile as required to protect vulnerable road users.

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