Fast screening of Bifidobacterium longum sublethal stress conditions in a novel two-stage continuous culture strategy

2013 ◽  
Vol 4 (2) ◽  
pp. 167-178 ◽  
Author(s):  
V. Mozzetti ◽  
F. Grattepanche ◽  
B. Berger ◽  
E. Rezzonico ◽  
F. Arigoni ◽  
...  
Keyword(s):  
Cell Viability ◽  
Continuous Culture ◽  
Bifidobacterium Longum ◽  
Food Additives ◽  
Stress Conditions ◽  
Stress Factors ◽  
Exponential Growth Phase ◽  
Two Stage ◽  
Batch Cultures ◽  
Sublethal Stress

A central issue in the application of probiotics as food additives is their fastidious production and their sensitivity to many environmental stresses. The importance of inducible cell-protective mechanisms triggered by application of sublethal stresses for survival under stress conditions has been demonstrated. Continuous cultures could be a suitable and more efficient method to test stress factors on one culture instead of several repeated batch cultures. In this study, the application of a two-stage continuous culture of Bifidobacterium longum NCC2705 was investigated. The first reactor was operated under fixed conditions at 37 °C and pH 6.0 and used to produce cells with controlled physiology, mimicking cells in the late exponential growth phase. Stress pretreatment combinations of pH (6.0, 5.0 and 4.0), temperature (37, 45 and 47 °C) and NaCl (0, 5 and 10%) were tested in the second reactor. Of all tested combinations, only those of pH 4.0 significantly decreased cell viability in the second reactor compared to control conditions (37 °C, pH 6.0, 0% NaCl) and, therefore, could not be considered as sublethal stresses. Pretreatments with 5 or 10% NaCl had a negative effect on cell viability after gastric lethal stress. A significant improvement in cell resistance to heat lethal stress (56 °C, 5 min) was observed for cells pretreated at 47 °C. In contrast, heat pretreatment negatively affected cell viability after freeze drying and osmotic lethal stresses. The two-stage continuous culture allowed for efficient screening of several stress pretreatments during the same experiment with up to four different conditions tested per day. Optimal sublethal stress conditions can also be applied for producing cells with traditional batch cultures.

Transcriptome analysis and physiology of Bifidobacterium longum NCC2705 cells under continuous culture conditions

2012 ◽  
Vol 3 (4) ◽  
pp. 261-272 ◽  
Author(s):  
V. Mozzetti ◽  
F. Grattepanche ◽  
D. Moine ◽  
B. Berger ◽  
E. Rezzonico ◽  
...  
Keyword(s):  
Continuous Culture ◽  
Bifidobacterium Longum ◽  
Viable Cell ◽  
Culture Conditions ◽  
Stress Conditions ◽  
Stress Factors ◽  
Bacterial Cells ◽  
Continuous Cultures ◽  
Batch Cultures ◽  
Cell Counts

A central issue in the use of probiotics in food and food supplements is their sensitivity to many environmental stress factors. The resistance of probiotic cells to lethal stress can be improved by application of homologous or heterologous sub-lethal stress during culture. This screening procedure is generally performed using batch cultures. Continuous cultures could be a suitable and more efficient method to test different stress factors on one culture instead of repeating several batch cultures. However, before testing stresses using continuous cultures, the physiological stability of continuously produced cells over a considered time period must be first evaluated. A continuous culture of Bifidobacterium longum NCC2705 was maintained for 211 h at a dilution rate of 0.1 per h, mimicking a deceleration growth phase culture. Stable viable cell counts were measured over the culture period, decreasing only moderately from 8.8 to 8.6 log10 cfu/ml. A slight shift in metabolite production, characterized by increased lactate and decreased acetate, formate and ethanol concentrations was observed. Susceptibilities to antibiotics and stress conditions were stable (cefotaxim, ampicillin, ceftazidime) or moderately affected (simulated gastric juices, heat, bile salts, tetracycline, chloramphenicol, penicillin, vancomycin and neomycin) over culturing time. Comparison of gene transcription profiles between samples collected after 31 h of continuous culture and samples collected after 134 and 211 h revealed only limited changes in expression of 1.0 and 3.8% of total genes, respectively. Based on these results, we propose that continuous culture can be used to produce bacterial cells with stable physiological properties suitable for fast and efficient screening of sub-lethal stress conditions.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals Knockdown of 60S Ribosomal Protein L14-2 Reveals Their Potential Regulatory Roles in Enhancing Drought and Salt Tolerance in Cotton

2021 ◽  
Author(s):  
Margaret shiraku ◽  
Richard Odongo Magwanga ◽  
Xiaoyan Cai ◽  
Joy Nyangasi Kirungu ◽  
Yanchao Xu ◽  
...  
Keyword(s):  
Salt Stress ◽  
Ribosomal Protein ◽  
Candidate Gene ◽  
Regulatory Elements ◽  
Stress Conditions ◽  
Stress Factors ◽  
Positive Effects ◽  
Drought And Salt Tolerance ◽  
Drought And Salt Stresses ◽  
Effects Of Drought

Abstract BackgroundCotton is an important economic crop and the primary source of natural fiber. The effects of drought and salt stresses threaten strong fiber and large quantity production. However, due to the ever-changing climatic conditions, plants have evolved various mechanisms to cope with the effects of various stress factors. One of the plant's transcription factors with positive effects in alleviating effects of drought and salt stresses is the Ribosomal protein Large (RPL) gene families. This has prompted the functional characterization of the RPL14B gene previously identified in the QTL region as a candidate gene that responds to stress and initiates mechanisms that enhance stress tolerance. ResultsComprehensive identification and functional analysis were conducted in this study, in which 26, 8, and 5 proteins containing the RPL14B domain were identified in G. hirsutum, G. raimondii, and G. arboreum, respectively. Moreover, Cis-regulatory elements associated with the RPL genes were identified. The Myb binding sites (MBS), Myb, Abscisic acid-responsive element (ABRE), CAAT-box, TATA box, TGACG-motif, and CGTCA-motif responsive to Meja, and TCA- motif responsive to salicylic acid were identified. Validation of the candidate gene through virus-induced gene silencing (VIGS) revealed that the Gh_D01G0234 (RPL14B) knockdown significantly affected the cotton seedling's performance under drought/ salt stress conditions as evidenced by a significant reduction in various morphological and physiological traits. Moreover, antioxidant enzyme levels were significantly reduced in VIGS-plants, with substantially higher oxidant enzyme levels, as evidenced by the higher concentration level of Malondialdehyde (MDA). ConclusionThe results revealed the potential role of the gene, and it can be further exploited to breed climate-smart cotton varieties resilient to drought and salt stress conditions

scholarly journals Colonization of Mucin by Human Intestinal Bacteria and Establishment of Biofilm Communities in a Two-Stage Continuous Culture System

2005 ◽  
Vol 71 (11) ◽  
pp. 7483-7492 ◽  
Author(s):  
Sandra Macfarlane ◽  
Emma J. Woodmansey ◽  
George T. Macfarlane
Keyword(s):  
Continuous Culture ◽  
Culture System ◽  
Laser Scanning ◽  
Intestinal Bacteria ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Culture Methods ◽  
Two Stage ◽  
Bacterial Populations ◽  
Continuous Culture System

ABSTRACT The human large intestine is covered with a protective mucus coating, which is heavily colonized by complex bacterial populations that are distinct from those in the gut lumen. Little is known of the composition and metabolic activities of these biofilms, although they are likely to play an important role in mucus breakdown. The aims of this study were to determine how intestinal bacteria colonize mucus and to study physiologic and enzymatic factors involved in the destruction of this glycoprotein. Colonization of mucin gels by fecal bacteria was studied in vitro, using a two-stage continuous culture system, simulating conditions of nutrient availability and limitation characteristic of the proximal (vessel 1) and distal (vessel 2) colon. The establishment of bacterial communities in mucin gels was investigated by selective culture methods, scanning electron microscopy, and confocal laser scanning microscopy, in association with fluorescently labeled 16S rRNA oligonucleotide probes. Gel samples were also taken for analysis of mucin-degrading enzymes and measurements of residual mucin sugars. Mucin gels were rapidly colonized by heterogeneous bacterial populations, especially members of the Bacteroides fragilis group, enterobacteria, and clostridia. Intestinal bacterial populations growing on mucin surfaces were shown to be phylogenetically and metabolically distinct from their planktonic counterparts.

Exponential growth phase ofPseudomonas methanolica batch cultures

1973 ◽  
Vol 13 (6) ◽  
pp. 523-528 ◽  
Author(s):  
E. M. Shulgovskaya ◽  
I. I. Ivanova ◽  
G. G. Sotnicov
Keyword(s):  
Growth Phase ◽  
Exponential Growth ◽  
Exponential Growth Phase ◽  
Batch Cultures

scholarly journals Regulation of gbpC Expression in Streptococcus mutans

2007 ◽  
Vol 189 (18) ◽  
pp. 6521-6531 ◽  
Author(s):  
Indranil Biswas ◽  
Laura Drake ◽  
Saswati Biswas
Keyword(s):  
Streptococcus Mutans ◽  
Dna Sequences ◽  
Promoter Region ◽  
Response Regulator ◽  
Surface Protein ◽  
Maximum Level ◽  
Stress Conditions ◽  
Pcr Analysis ◽  
Exponential Growth Phase ◽  
Transcriptional Level

ABSTRACT Streptococcus mutans, the principal causative agent of dental caries, produces four glucan-binding proteins (Gbp) that play major roles in bacterial adherence and pathogenesis. One of these proteins, GbpC, is an important cell surface protein involved in biofilm formation. GbpC is also important for cariogenesis, bacteremia, and infective endocarditis. In this study, we examined the regulation of gbpC expression in S. mutans strain UA159. We found that gbpC expression attains the maximum level at mid-exponential growth phase, and the half-life of the transcript is less than 2 min. Expression from PgbpC was measured using a PgbpC-gusA transcriptional fusion reporter and was analyzed under various stress conditions, including thermal, osmotic, and acid stresses. Expression of gbpC is induced under conditions of thermal stress but is repressed during growth at low pH, whereas osmotic stress had no effect on expression from PgbpC. The results from the expression analyses were further confirmed using semiquantitative reverse transcription-PCR analysis. Our results also reveal that CovR, a global response regulator in many Streptococcus spp., represses gbpC expression at the transcriptional level. We demonstrated that purified CovR protein binds directly to the promoter region of PgbpC to repress gbpC expression. Using a DNase I protection assay, we showed that CovR binds to DNA sequences surrounding PgbpC from bases −68 to 28 (where base 1 is the start of transcription). In summary, our results indicate that various stress conditions modulate the expression of gbpC and that CovR negatively regulates the expression of the gbpC gene by directly binding to the promoter region.

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