scholarly journals Cannibalism as cell differentiation meant that Bacillus subtilis NRS-762 is not suitable as model organism in survivability studies of microbes

2017 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Cell Population ◽  
Optical Density ◽  
Model Organism ◽  
Cell Lysis ◽  
Model Organisms ◽  
Rapid Decline ◽  
Post Inoculation ◽  
Massive Cell

Survival of microbes on various surfaces and environment is a question of importance to basic science, as well as health care, water treatment and distribution, ecology, and search for life in other planetary bodies. To this end, various model organisms, known to be resilient against a variety of environmental insults are used for understanding the mechanisms underlying survival in extreme environments, or conditions mimicking those of the investigated habitats. Serendipitous observations of drastic decline in optical density of Bacillus subtilis NRS-762 (ATCC 8473) in LB Lennox and Tryptic Soy Broth (TSB) at temperatures of 25, 30 and 37 oC, after the aerobic culture reached maximal cell density at stationary phase, pointed to possible cell lysis as mechanism for cell death. Specifically, optical density of the bacterium declined from 5.4 at 22.5 hours post inoculation in LB Lennox to 2.5 after 38 hours of culture at 25 oC and 250 rpm rotational shaking. Similarly, optical density of B. subtilis also precipitously declined from 6.4 at 33 hours of culture to 1.8 at 51 hours post inoculation at 37 oC in TSB. This is in stark contrast to aerobic growth of Escherichia coli DH5α (ATCC 53868) in LB Lennox at 37 oC and 250 rpm, where optical density remained stable during stationary phase. More importantly, observations of B. subtilis culture after autoclave decontamination revealed lack of cellular debris; thereby, indicating massive cell lysis resulting in population collapse. Although B. subtilis is known to enter into various cellular differentiation programmes upon nutrient starvation such as onset of stationary phase in cell culture, complete absence of cell debris that usually settle at the bottom of the shake flask after autoclave decontamination, pointed to cannibalism or prophage induced cell lysis as key reasons underlying observed drastic decline in optical density of the culture. Specifically, prophage induced cell lysis may be discounted as this would have destroyed the entire cell population expeditiously shortly after entry into stationary phase. Hence, cannibalism, where a subpopulation of B. subtilis cells secrete cell lysis factors which other B. subtilis cells are not resistant to, likely result in massive cell lysis that generated cellular contents that could serve as nutrients for the surviving cell population resistant to the cell lysis factors, and may be the dominant mechanism underpinning observed rapid decline in optical density after entry into stationary phase. Collectively, B. subtilis NRS-762 is not suitable as model organism for microbial survivability studies given its tendency to undergo differentiation into the cannibalism programme, which in killing a significant fraction of cells upon nutrient deprivation, would also confound experiments aimed at understanding the resilience of cells towards various extraneous environmental factors not common in the microbe’s favoured habitats.

scholarly journals Drastic decline in optical density during stationary phase meant that Bacillus subtilis NRS-762 is not suitable as model organism in microbial survivability studies

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Optical Density ◽  
Shake Flask ◽  
Model Organism ◽  
Cell Lysis ◽  
Nutrient Starvation ◽  
Model Organisms ◽  
Post Inoculation ◽  
Massive Cell

Survival of microbes on various surfaces and habitats is a question of importance to basic science, as well as health care, water treatment and distribution, ecology, and search for life in other planetary bodies. To this end, various model organisms known to be resilient against a variety of environmental stressors are used for understanding the mechanisms underlying survival in extreme environments, or conditions mimicking those of the investigated habitats. Observations of drastic decline in optical density of Bacillus subtilis NRS-762 (ATCC 8473) in LB Lennox and Tryptic Soy Broth (TSB) at temperatures of 25, 30 and 37 oC, after the aerobic shake flask culture reached maximal cell density at stationary phase, pointed to possible cell lysis as mechanism for cell death. Specifically, optical density of the bacterium declined from 5.4 at 22.5 hours post-inoculation in LB Lennox medium to 2.5 after 38 hours of culture at 25 oC and 250 rpm rotational shaking. Similarly, optical density of B. subtilis NRS-762 also precipitously declined from 6.4 at 33 hours of culture to 1.8 at 51 hours post-inoculation at 37 oC in TSB. This is in stark contrast to aerobic growth of Escherichia coli DH5α (ATCC 53868) in LB Lennox medium at 37 oC and 230 rpm rotational shaking, where optical density remained stable during stationary phase. More importantly, observations of B. subtilis NRS-762 culture after autoclave decontamination revealed a lack of cellular debris; thereby, indicating massive cell lysis resulting in population collapse. Although B. subtilis is known to enter into various cellular differentiation programmes upon nutrient starvation, complete absence of cell debris that usually settle at the bottom of the shake flask after autoclave decontamination pointed to cannibalism or prophage induced cell lysis as key reasons underlying observed drastic decline in optical density of the culture. However, prophage induced cell lysis may be discounted as this would have led to rapid collapse of the entire cell population shortly after entry into stationary phase except during growth of B. subtilis NRS-762 at 37 oC where a temperature sensitive sensor might have activated prophage entry into the lytic programme. Hence, cannibalism, where a subpopulation of B. subtilis NRS-762 cells secrete cell lysis factors which other B. subtilis NRS-762 cells are not resistant to, likely resulted in massive cell lysis that released cellular contents that served as nutrients for the surviving population. Collectively, B. subtilis NRS-762 is not suitable as model organism for microbial survivability studies given its tendency to undergo differentiation into the cannibalism programme, which in killing a significant fraction of cells upon nutrient starvation, would confound experiments aimed at understanding the survivability of the bacterium under a variety of environmental conditions.

scholarly journals Drastic decline in optical density during stationary phase meant that Bacillus subtilis NRS-762 is not suitable as model organism in microbial survivability studies

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Optical Density ◽  
Shake Flask ◽  
Model Organism ◽  
Cell Lysis ◽  
Nutrient Starvation ◽  
Model Organisms ◽  
Post Inoculation ◽  
Massive Cell

Survival of microbes on various surfaces and habitats is a question of importance to basic science, as well as health care, water treatment and distribution, ecology, and search for life in other planetary bodies. To this end, various model organisms known to be resilient against a variety of environmental stressors are used for understanding the mechanisms underlying survival in extreme environments, or conditions mimicking those of the investigated habitats. Observations of drastic decline in optical density of Bacillus subtilis NRS-762 (ATCC 8473) in LB Lennox and Tryptic Soy Broth (TSB) at temperatures of 25, 30 and 37 oC, after the aerobic shake flask culture reached maximal cell density at stationary phase, pointed to possible cell lysis as mechanism for cell death. Specifically, optical density of the bacterium declined from 5.4 at 22.5 hours post-inoculation in LB Lennox medium to 2.5 after 38 hours of culture at 25 oC and 250 rpm rotational shaking. Similarly, optical density of B. subtilis NRS-762 also precipitously declined from 6.4 at 33 hours of culture to 1.8 at 51 hours post-inoculation at 37 oC in TSB. This is in stark contrast to aerobic growth of Escherichia coli DH5α (ATCC 53868) in LB Lennox medium at 37 oC and 230 rpm rotational shaking, where optical density remained stable during stationary phase. More importantly, observations of B. subtilis NRS-762 culture after autoclave decontamination revealed a lack of cellular debris; thereby, indicating massive cell lysis resulting in population collapse. Although B. subtilis is known to enter into various cellular differentiation programmes upon nutrient starvation, complete absence of cell debris that usually settle at the bottom of the shake flask after autoclave decontamination pointed to cannibalism or prophage induced cell lysis as key reasons underlying observed drastic decline in optical density of the culture. However, prophage induced cell lysis may be discounted as this would have led to rapid collapse of the entire cell population shortly after entry into stationary phase except during growth of B. subtilis NRS-762 at 37 oC where a temperature sensitive sensor might have activated prophage entry into the lytic programme. Hence, cannibalism, where a subpopulation of B. subtilis NRS-762 cells secrete cell lysis factors which other B. subtilis NRS-762 cells are not resistant to, likely resulted in massive cell lysis that released cellular contents that served as nutrients for the surviving population. Collectively, B. subtilis NRS-762 is not suitable as model organism for microbial survivability studies given its tendency to undergo differentiation into the cannibalism programme, which in killing a significant fraction of cells upon nutrient starvation, would confound experiments aimed at understanding the survivability of the bacterium under a variety of environmental conditions.

scholarly journals Development of Leishmania (Mundinia) in guinea pigs

2019 ◽  
Author(s):  
Tomáš Bečvář ◽  
Padet Siriyasatien ◽  
Paul Bates ◽  
Petr Volf ◽  
Jovana Sádlová
Keyword(s):  
Guinea Pigs ◽  
Model Organism ◽  
Model Organisms ◽  
Control Group ◽  
Post Inoculation ◽  
Sand Flies ◽  
Tissue Samples ◽  
Wild Mammals ◽  
Reservoir Hosts ◽  
Lutzomyia Migonei

Abstract BackgroundLeishmaniasis is a human and animal disease caused by parasites of the genus Leishmania, which is now divided into 4 subgenera – L. (Leishmania), L. (Viannia), L. (Sauroleishmania) and L. (Mundinia). Subgenus Mundinia, established in 2016, is geographically widely dispersed, its distribution covers all continents, except Antarctica. It consists of 5 species - L. enriettii and L. macropodum are parasites of wild mammals while L. martiniquensis, L. orientalis and unnamed L. sp. from Ghana are infectious to humans. There is very little information on natural reservoir hosts and vectors for any Mundinia species. MethodsExperimental infections of guinea-pigs with all five Mundinia species were performed. Animals were injected intradermally with 107 culture-derived promastigotes into both ear pinnae. The courses of infections were monitored weakly; xenodiagnoses were performed at weeks 4 and 8 post infection using Lutzomyia migonei. The distribution of parasites in different tissues was determined post mortem by conventional PCR.ResultsNo significant differences in weight were observed between infected animals and the control group. Animals infected with L. enriettii developed temporary lesions at the site of inoculation and were infectious to Lu. migonei in xenodiagnoses. Animals infected with L. martiniquensis and L. orientalis developed temporary erythema and dry lesions at the site of inoculation, respectively, but were not infectious to sand flies. Guinea pigs infected by L. macropodum and L. sp. from Ghana showed no signs of infection during experiments, were not infectious to sand flies and leishmanial DNA was not detected in their tissue samples at the end of experiments at week 12 post-inoculation.ConclusionsAccording to our results, guinea pigs are not an appropriate model organism for studying Mundinia species other than L. enriettii. We suggest that for better understanding of L. (Mundinia) biology it is necessary to focus on other model organisms.

scholarly journals Nonclassical Protein Secretion by Bacillus subtilis in the Stationary Phase Is Not Due to Cell Lysis

2011 ◽  
Vol 193 (20) ◽  
pp. 5607-5615 ◽  
Author(s):  
C.-K. Yang ◽  
H. E. Ewis ◽  
X. Zhang ◽  
C.-D. Lu ◽  
H.-J. Hu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Protein Secretion ◽  
Cell Lysis

scholarly journals A Modification of Cell-Lysis for High-Quality Intact RNA of Bacillus Subtilis Extracted From the Culture Under Different Physiological Stages

2021 ◽  
Author(s):  
Phetcharat Jaiaue ◽  
Piroonporn Srimong ◽  
Sitanan Thitiprasert ◽  
Somboon Tanasupawat ◽  
Benjamas Cheirsilp ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rna Extraction ◽  
Model Organism ◽  
Cell Lysis ◽  
Protein Precipitation ◽  
Absolute Ethanol ◽  
Process Conditions ◽  
High Quality ◽  
Gram Positive ◽  
Gram Positive Bacteria

Abstract High quality RNA products from bacterial cells are required for the molecular study. Sample preparation to acquire the high-quality RNA especially the Gram-positive bacteria like Bacillus sp., the model organism, remains a critical burden toward the integration of full molecular downstream analyses although several methods have been proposed including conventional or kit-based protocols. Those techniques were simply developed using the cell samples at certain growth stages unless some molecular studies require RNA samples gathered under different physiological stages of growth and process conditions. Herein, we developed the simple yet effective cell-lysis technique prior to RNA extraction by modifying the commercial kit-based protocols. Bacillus subtilis TL7-3 was used as the model organism in this study. Lysozyme loading (20 mg/mL) as well as the incubation time (30 min) and temperature (37 °C) was responsible for cell lysis and increased RNA concentration in the samples. Invert mixing rather than centrifugation and vortexing prevented RNA damage during protein precipitation by absolute ethanol. This was confirmed by the RNA Integrity Number (RIN) values greater than 8.0 of all RNA extracted from both vegetative cells and endospores of B. subtilis TL7-3. Additionally, absolute ethanol is preferable to our less-than-1-h protocol for protein precipitation as indicated from the higher ratios of A260/A280 and those of A260/A230 of the RNA products than 2.0 and 2.1, respectively. From the findings mentioned above, we successfully developed the modified RNA extraction protocol applicable for the intact cells of Gram-positive bacteria like Bacillus sp. at varied physiological and morphological stages.

scholarly journals Biosurfactant production maintains viability in anoxic conditions by depolarizing the membrane in Bacillus subtilis

2019 ◽  
Author(s):  
Heidi A. Arjes ◽  
Lam Vo ◽  
Caroline Marie Dunn ◽  
Lisa Willis ◽  
Christopher A. DeRosa ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Membrane Potential ◽  
Stationary Phase ◽  
Optical Density ◽  
Electron Acceptor ◽  
Oxygen Depletion ◽  
Aerobic Growth ◽  
Terminal Electron Acceptor ◽  
Obligate Aerobe

SummaryThe presence or absence of oxygen in the environment is a strong effector of cellular metabolism and physiology. Like many eukaryotes and some bacteria, Bacillus subtilis is an obligate aerobe that primarily utilizes oxygen during respiration to generate ATP. Despite the importance of oxygen for B. subtilis survival, we know little about how oxygen is consumed during growth and how populations respond to shifts in oxygen availability. Here, we find that when oxygen was depleted from stationary phase cultures ∼90% of B. subtilis 3610 cells died and lysed due to autolysin activity; the remaining cells maintained colony-forming ability. Interestingly, the domesticated 168 strain maintained a higher optical density than 3610 during oxygen depletion due to the formation of cell-wall-less protoplasts, but the remaining, rod-shaped cells were >100-fold less viable than 3610. We discovered that the higher viability in 3610 was due to its ability to produce the antibacterial compound surfactin, as surfactin addition rescued 168 viability and also increased yield in aerobic growth. We further demonstrate that surfactin strongly depolarizes the B. subtilis membrane, and that other known membrane-potential disruptors restore viability to 168. These findings highlight the importance of surfactin for survival during oxygen-depleted conditions and demonstrate that antimicrobials normally considered harmful can instead benefit cells in stressful conditions when the terminal electron acceptor in respiration is limiting.

scholarly journals Development of Leishmania (Mundinia) in guinea pigs

2020 ◽  
Author(s):  
Tomáš Bečvář ◽  
Padet Siriyasatien ◽  
Paul Bates ◽  
Petr Volf ◽  
Jovana Sádlová
Keyword(s):  
Guinea Pigs ◽  
Model Organism ◽  
Model Organisms ◽  
Control Group ◽  
Post Inoculation ◽  
Sand Flies ◽  
Tissue Samples ◽  
Wild Mammals ◽  
Reservoir Hosts ◽  
Lutzomyia Migonei

Abstract Background: Leishmaniasis is a human and animal disease caused by parasites of the genus Leishmania , which is now divided into 4 subgenera – L. (Leishmania) , L. (Viannia) , L. (Sauroleishmania) and L. (Mundinia) . Subgenus Mundinia, established in 2016, is geographically widely dispersed, its distribution covers all continents, except Antarctica. It consists of 5 species - L. enriettii and L. macropodum are parasites of wild mammals while L. martiniquensis , L. orientalis and unnamed L . sp. from Ghana are infectious to humans. There is very little information on natural reservoir hosts and vectors for any Mundinia species. Methods: Experimental infections of guinea-pigs with all five Mundinia species were performed. Animals were injected intradermally with 10 7 culture-derived promastigotes into both ear pinnae. The courses of infections were monitored weekly; xenodiagnoses were performed at weeks 4 and 8 post infection using Lutzomyia migonei . The distribution of parasites in different tissues was determined post mortem by conventional PCR. Results: No significant differences in weight were observed between infected animals and the control group. Animals infected with L. enriettii developed temporary lesions at the site of inoculation and were infectious to Lu. migonei in xenodiagnoses. Animals infected with L. martiniquensis and L. orientalis developed temporary erythema and dry lesions at the site of inoculation, respectively, but were not infectious to sand flies. Guinea pigs infected by L. macropodum and L . sp . from Ghana showed no signs of infection during experiments, were not infectious to sand flies and leishmanial DNA was not detected in their tissue samples at the end of experiments at week 12 post-inoculation. Conclusions: According to our results, guinea pigs are not an appropriate model organism for studying Mundinia species other than L. enriettii. We suggest that for better understanding of L. (Mundinia) biology it is necessary to focus on other model organisms.

scholarly journals Development of Leishmania (Mundinia) in guinea pigs

2020 ◽  
Author(s):  
Tomáš Bečvář ◽  
Padet Siriyasatien ◽  
Paul Bates ◽  
Petr Volf ◽  
Jovana Sádlová
Keyword(s):  
Guinea Pigs ◽  
Model Organism ◽  
Model Organisms ◽  
Control Group ◽  
Post Inoculation ◽  
Sand Flies ◽  
Tissue Samples ◽  
Wild Mammals ◽  
Leishmania Enriettii ◽  
Reservoir Hosts

Abstract Background: Leishmaniasis is a human and animal disease caused by parasites of the genus Leishmania , which is now divided into 4 subgenera – L. (Leishmania) , L. (Viannia) , L. (Sauroleishmania) and L. (Mundinia) . Subgenus Mundinia, established in 2016, is geographically widely dispersed, its distribution covers all continents, except Antarctica. It consists of 5 species - L. enriettii and L. macropodum are parasites of wild mammals while L. martiniquensis , L. orientalis and unnamed L . sp. from Ghana are infectious to humans. There is very little information on natural reservoir hosts and vectors for any Mundinia species. Methods: Experimental infections of guinea-pigs with all five Mundinia species were performed. Animals were injected intradermally with 10 7 culture-derived promastigotes into both ear pinnae. The courses of infections were monitored weekly; xenodiagnoses were performed at weeks 4 and 8 post infection using Lutzomyia migonei . The distribution of parasites in different tissues was determined post mortem by conventional PCR. Results: No significant differences in weight were observed between infected animals and the control group. Animals infected with L. enriettii developed temporary lesions at the site of inoculation and were infectious to Lu. migonei in xenodiagnoses. Animals infected with L. martiniquensis and L. orientalis developed temporary erythema and dry lesions at the site of inoculation, respectively, but were not infectious to sand flies. Guinea pigs infected by L. macropodum and L . sp . from Ghana showed no signs of infection during experiments, were not infectious to sand flies and leishmanial DNA was not detected in their tissue samples at the end of experiments at week 12 post-inoculation. Conclusions: According to our results, guinea pigs are not an appropriate model organism for studying Mundinia species other than L. enriettii. We suggest that for better understanding of L. (Mundinia) biology it is necessary to focus on other model organisms. Key words: Leishmania, Mundinia , guinea pig, Leishmania enriettii, Leishmania martiniquensis, Leishmania orientalis, Leishmania macropodum, animal model

Ultrastructural Changes in the Mammary Epithelial Cell Population During Neoplastic Development Induced by A Chemical Carcinogen.

1976 ◽  
Vol 34 ◽  
pp. 250-251 ◽  
Author(s):  
J. Russo ◽  
W. Isenberg ◽  
M. Ireland ◽  
I.H. Russo
Keyword(s):  
Mammary Gland ◽  
Cell Population ◽  
Virgin Female ◽  
Histological Change ◽  
Mammary Epithelial ◽  
Female Rats ◽  
Ultrastructural Changes ◽  
Post Inoculation ◽  
Chemical Carcinogen ◽  
Sprague Dawley

The induction of rat mammary carcinoma by the chemical carcinogen DMBA is used as a model for the study of the human disease (1). We previously described the histochemical changes that occur in the mammary gland of DMBA treated animals before the earliest manifested histological change, the intraductal proliferation (IDP), was observed (2). In the present work, we demonstrate that a change in the stable cell population found in the resting mammary gland occurs after carcinogen administration.Fifty-five day old Sprague-Dawley virgin female rats were inoculated intragastrically with 20mg of 7,12-dimethylbenz(a)anthracene (DMBA) in 1ml sesame oil. Non-inoculated, age-matched females were used as controls. Mammary glands from control and inoculated rats were removed weekly from the time of inoculation until 60 days post-inoculation. For electron microscopy, the glands were immersed in Karnovsky's fixative, post-fixed in 1% OsO4, dehydrated, and embedded in an Epon-Araldite mixture. Thick (lμ) sections were stained with 1% toluidine blue and were used for selecting areas for ultrastructural study.

scholarly journals In Search of Species-Specific SNPs in a Non-Model Animal (European Bison (Bison bonasus))—Comparison of De Novo and Reference-Based Integrated Pipeline of STACKS Using Genotyping-by-Sequencing (GBS) Data

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2226
Author(s):  
Sazia Kunvar ◽  
Sylwia Czarnomska ◽  
Cino Pertoldi ◽  
Małgorzata Tokarska
Keyword(s):  
Reference Genome ◽  
De Novo ◽  
Bos Taurus ◽  
Model Organism ◽  
Genotyping By Sequencing ◽  
Model Organisms ◽  
European Bison ◽  
Model Animal ◽  
Pcr Duplicates ◽  
Species Specific

The European bison is a non-model organism; thus, most of its genetic and genomic analyses have been performed using cattle-specific resources, such as BovineSNP50 BeadChip or Illumina Bovine 800 K HD Bead Chip. The problem with non-specific tools is the potential loss of evolutionary diversified information (ascertainment bias) and species-specific markers. Here, we have used a genotyping-by-sequencing (GBS) approach for genotyping 256 samples from the European bison population in Bialowieza Forest (Poland) and performed an analysis using two integrated pipelines of the STACKS software: one is de novo (without reference genome) and the other is a reference pipeline (with reference genome). Moreover, we used a reference pipeline with two different genomes, i.e., Bos taurus and European bison. Genotyping by sequencing (GBS) is a useful tool for SNP genotyping in non-model organisms due to its cost effectiveness. Our results support GBS with a reference pipeline without PCR duplicates as a powerful approach for studying the population structure and genotyping data of non-model organisms. We found more polymorphic markers in the reference pipeline in comparison to the de novo pipeline. The decreased number of SNPs from the de novo pipeline could be due to the extremely low level of heterozygosity in European bison. It has been confirmed that all the de novo/Bos taurus and Bos taurus reference pipeline obtained SNPs were unique and not included in 800 K BovineHD BeadChip.

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