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Exponential Growth Phase (exponential + growth_phase)
Selected AbstractsExistence of a tightly regulated water channel in Saccharomyces cerevisiaeFEBS JOURNAL, Issue 2 2001Valérie Meyrial The Saccharomyces cerevisiae strain ,1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p. Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2-1p in this system. Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast. Aqy1-1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2-1p expression was repressed by hyper-osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane. In microsomal vesicles enriched in ER, a water channel activity due to Aqy2-1p was detected by stopped-flow analysis. Our results show that the expression of aquaporins is tightly controlled. The physiological relevance of aquaporin-mediated water transport in yeast is discussed. [source] Characterisation of preYvaY export reveals differences in the substrate specificities of Bacillus subtilis and Escherichia coli leader peptidasesFEMS MICROBIOLOGY LETTERS, Issue 1 2003Dirk Linde Abstract Translocation, processing and secretion of YvaY, a Bacillus subtilis protein of unknown function, were characterised both in B. subtilis and in Escherichia coli. In its natural host B. subtilis, YvaY was transiently synthesised at the end of the exponential growth phase. It was efficiently secreted into the culture supernatant in spite of a calculated membrane spanning domain in the mature part of the protein. In E. coli, despite the high conservation of Sec-dependent transport components, processing of preYvaY was strongly impaired. To uncover which elements of E. coli and B. subtilis translocation systems are responsible for the observed substrate specificity, components of the B. subtilis Sec-system were co-expressed besides yvaY in E. coli. Expression of B. subtilis secA or secYEG genes did not affect processing, but expression of B. subtilis signal peptidase genes significantly enhanced processing of preYvaY in E. coli. While the major signal peptidases SipS or SipT had a strong stimulatory effect on preYvaY processing, the minor signal peptidases SipU, SipV or SipW had a far less stimulatory effect in E. coli. These results reveal that targeting and translocation of preYvaY is mediated by the E. coli Sec proteins but processing of preYvaY is not performed by E. coli signal peptidase LepB. Thus, differences in substrate specificities of E. coli LepB and the B. subtilis Sip proteins provide the bottleneck for export of YvaY in E. coli. Significant slower processing of preYvaY in absence of SecB indicated that SecB mediates targeting of the B. subtilis precursor. [source] Experimental silicification of the extremophilic Archaea Pyrococcus abyssi and Methanocaldococcus jannaschii: applications in the search for evidence of life in early Earth and extraterrestrial rocksGEOBIOLOGY, Issue 4 2009F. ORANGE Hydrothermal activity was common on the early Earth and associated micro-organisms would most likely have included thermophilic to hyperthermophilic species. 3.5,3.3 billion-year-old, hydrothermally influenced rocks contain silicified microbial mats and colonies that must have been bathed in warm to hot hydrothermal emanations. Could they represent thermophilic or hyperthermophilic micro-organisms and if so, how were they preserved? We present the results of an experiment to silicify anaerobic, hyperthermophilic micro-organisms from the Archaea Domain Pyrococcus abyssi and Methanocaldococcus jannaschii, that could have lived on the early Earth. The micro-organisms were placed in a silica-saturated medium for periods up to 1 year. Pyrococcus abyssi cells were fossilized but the M. jannaschii cells lysed naturally after the exponential growth phase, apart from a few cells and cell remains, and were not silicified although their extracellular polymeric substances were. In this first simulated fossilization of archaeal strains, our results suggest that differences between species have a strong influence on the potential for different micro-organisms to be preserved by fossilization and that those found in the fossil record represent probably only a part of the original diversity. Our results have important consequences for biosignatures in hydrothermal or hydrothermally influenced deposits on Earth, as well as on early Mars, as environmental conditions were similar on the young terrestrial planets and traces of early Martian life may have been similarly preserved as silicified microfossils. [source] Responses of gas exchange and growth in Merkus pine seedlings to expected climatic changes in ThailandGLOBAL CHANGE BIOLOGY, Issue 6 2001Jarkko Koskela Abstract Responses of gas exchange and growth in Merkus pine (Pinus merkusii Jungh. et de Vriese) seedlings to changing climate were analysed for high- and low-altitude sites in Thailand. A gas exchange model, based on the optimality approach, derived the effect of drought from the probability of rains. A carbon-and nitrogen-balance growth model applied structural regularities of a tree and a modification of functional balance between foliage and fine roots as growth- guiding rules. Adaptation to local climates was incorporated in the models. The simulations yielded physiologically reasonable behaviour for annual photosynthesis (A) and transpiration (E) in relation to the distributions of precipitation over the course of a year. An annual temperature increase of 2 °C and a prolonged dry season (scenario 2) reduced A by 5,11% and E by 5,8% as compared to present climate (scenario 1). Doubled CO2 concentration and the increased temperature (scenario 3) enhanced A by 56,59% and E by 14%. Simultaneously these changes (scenario 4) increased A by 41,53% and E by 1,5%. Simulated growth in scenario 1 fitted reasonably well to field data. By the age of five years, simulated total biomass (TB) and height (h) were reduced by 31,67% and 12,42%, respectively, in scenario 2 compared to scenario 1. In scenario 3, TB and h increased by 279,330% and 94,191%, and in scenario 4, by 83,241% and 55,69%, respectively. Large increases in TB and h are explained by the exponential growth phase of the young seedlings. These results suggest that climatic changes enhance growth and thus shorten the duration of the grass stage in these seedlings. However, the effects of climatic changes on growth depend strongly on how rainfall seasonality is altered in SE Asia because prolonged drought episodes may retard the fertilizing effects of the increasing CO2 concentration. [source] Vegetative growth and development of irrigated forage turnip (Brassica rapa var. rapa)GRASS & FORAGE SCIENCE, Issue 4 2008J. E. Neilsen Abstract Field and greenhouse experiments were conducted to identify visual markers and predictors of changes in the vegetative growth rate of forage turnip (Brassica rapa var. rapa) as a potential tool to improve the timing of inputs of N and irrigation to periods of maximum demand. The onset of root expansion, which was associated with a colour change and the death of cotyledons, was identified as a critical marker for the beginning of the rapid growth of the crop and the accumulation of starch in the storage root but indicators of subsequent changes in vegetative growth rate were not identifiable. The results suggested that management inputs can be more readily targeted to the beginning of the exponential growth phase but targeting of later vegetative growth stages will remain arbitrary. The vegetative growth and development of the crop was also studied to elucidate the process of leaf emergence and senescence (turnover) as they affected both leaf and root yield. The sequential senescence of leaves, which began immediately after cotyledon death, and translocation of carbohydrate to the storage root, coupled with high leaf area index (LAI), probably account for the high growth rates of 220 kg ha,1 day,1 maintained for periods of 10 weeks after the onset of root expansion. High yields can be expected if high LAI is maintained by ensuring that leaf emergence rates are not limited by nutrient or water deficiencies and leaves are protected from insect pests. Forage turnip is particularly robust because new leaf continues to emerge as older and damaged leaves senesce and carbohydrate is stored as starch in the storage root. [source] Beneficial links for the control of aphids: the effects of compost applications on predators and preyJOURNAL OF APPLIED ECOLOGY, Issue 4 2008James R. Bell Summary 1Polyphagous predators, such as spiders and beetles, perform a fundamental ecosystem service as regulators of agricultural pests, particularly aphids. They are most effective when they colonize the crop before the pest has reached its exponential growth phase. However, this is also when predators find themselves in a state of near-starvation. 2Predator numbers can be enhanced by applications of different types of organic matter, but the mechanism is not clearly understood. One hypothesis is that compost applied to the field may introduce a new detrital food chain to maintain predators until the pest arrives, but this may also be detrimental to effective pest control, fostering a surplus of alternative prey and causing a switch away from the pest. To elucidate these possible outcomes, we report on the use of within-field compost applications on aphids and their predators, presenting 4 years of field-scale manipulations. 3We found both direct and indirect links between compost, aphids and predators. In years when compost-treated plots had significantly higher numbers of predators, aphids were in significantly lower numbers than in plots without compost. Conversely, when there was a lack of response by predators, aphid numbers showed similar trends in all treatments. 4In all years, alternative prey responded strongly to compost application and did not fluctuate at the level shown by predators, suggesting that these two prey groups were decoupled. Instead, the predicted positive feedback of compost on predators numbers was either weak or absent. 5Synthesis and applications. The effect of compost on aphids clearly requires further practical refinement if it is to provide constant pest suppression, making it difficult to provide specific management recommendations at this stage. In the short term, compost application may not always confer immediate benefits in terms of pest control alone but this must be set against other better known benefits (moisture retention, nutrients). In the long term, experiments measuring the full trophic pathway are needed to unravel the effects of organic matter type, application time and the siting of compost relative to the crop in order to optimise pest suppression potential. [source] Pathways that produce volatile sulphur compounds from methionine in Oenococcus oeniJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2008A. Vallet Abstract Aims:, Determination of pathways involved in synthesis of volatile sulphur compounds (VSC) from methionine by Oenococcus oeni isolated from wine. Methods and Results:, Production of VSC by O. oeni from methionine was investigated during bacterial cultures and in assays performed in the presence of resting cells or protein fractions. Cells of O. oeni grown in a medium supplemented with methionine produced methanethiol, dimethyl disulphide, methionol and 3-(methylthio)propionic acid. Methional was also detected, but only transiently during the exponential growth phase. It was converted to methionol and 3-(methylthio) propionic acid in assays. Although this acid could be produced alternatively from 2-oxo-4-(methylthio) butyric acid (KMBA) by oxidative decarboxylation. In addition, KMBA was a precursor for methanethiol and dimethyl disulphide synthesis. Interestingly, assays with resting cells and protein fractions suggested that a specific enzyme could be involved in this conversion in O. oeni. Conclusion:, This work shows that methional and KMBA are the key intermediates for VSC synthesis from methionine in O. oeni. Putative enzymatic and chemical pathways responsible for the production of these VSC are discussed. Significance and impact of the study:, This work confirms the capacity of O. oeni to metabolize methionine and describes the involvement of potential enzymatic pathways. [source] Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperaturesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2004E. van Rensburg Abstract Aims:, To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. Methods and Results:,Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, ,18 and ,75°C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain,heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. Conclusions:,Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. Significance and Impact of the Study:, Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen. [source] Exopolysaccharide (EPS) biosynthesis by Lactobacillus sakei 0,1: production kinetics, enzyme activities and EPS yieldsJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2001B. Degeest Aims:,To determine optimal exopolysaccharide (EPS) production conditions of the mesophilic lactic acid bacterium strain Lactobacillus sakei 0,1 and to detect possible links between EPS yields and the activity of relevant enzymes. Methods and Results:,Fermentation experiments at different temperatures using either glucose or lactose were carried out. EPS production took place during the exponential growth phase. Low temperatures, applying glucose as carbohydrate source, resulted in the best bacterial growth, the highest amounts of EPS and the highest specific EPS production. Activities of 10 important enzymes involved in the EPS biosynthesis and the energy formation of Lact. sakei 0,1 were measured. The obtained results revealed that there is a clear link for some enzymes with EPS biosynthesis. It was also demonstrated clearly that the presence of rhamnose in the EPS building blocks is due to high activities of the enzymes involved in the rhamnose synthetic branch. Conclusions:,EPS production in Lact. sakei 0,1 is growth-associated and displays primary metabolite kinetics. Glucose as carbohydrate source and low temperatures enhance the EPS production. The enzymes involved in the biosynthesis of the activated sugar nucleotides play a major role in determining the monomeric composition of the synthesized EPS. Significance and Impact of the Study:,The proposed results contribute to a better understanding of the physiological factors influencing EPS production and the key enzymes involved in EPS biosynthesis by Lact. sakei. [source] Carbon monoxide and oxidative stress in Desulfovibrio desulfuricans B-1388JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2004M. Davydova Abstract It has been shown that carbon monoxide (CO) in low concentration may be an active biochemical and physiological regulator of cell function. The bases of CO toxicity and cell protection are not clearly understood. To provide insights into these mechanisms, we measured superoxide production by D. desulfuricans B-1388 incubated anaerobically in Postgate medium with or without 5% CO. D. desulfuricans B-1388 growing with CO in the gas phase produced more superoxide radicals then control cells growing in Ar. When the cells were pregrown with CO, NADH oxidase and peroxidase activities were increased. The increase in peroxidase activities of cells growing under CO (particularly NADH peroxidase) suggested that H2O2 was accumulated in cells. Superoxide dismutase (SOD) activity of cells decreased in exponential growth phase and increased in stationary phase. This may be due to CO concentration fall during CO oxidation by CO dehydrogenase. Altogether, our data suggest that superoxide production is a possible mechanism of CO toxicity. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:87,91, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20011 [source] GENERATION OF BIOLUMINESCENT MORGANELLA MORGANII AND ITS POTENTIAL USAGE IN DETERMINATION OF GROWTH LIMITS AND HISTAMINE PRODUCTIONJOURNAL OF FOOD SAFETY, Issue 2 2009MEHDI ZAREI ABSTRACT A mini-Tn5 promoter probe carrying the intact lux operon of Photorhabdus luminescens (pUT mini-Tn5 luxCDABE) which allowed measurement of light output without the addition of exogenous substrate was constructed. It was used to create a pool of chromosomally lux -marked strains of Morganella morganii. Also plasmid-mediated expression of bioluminescence in M. morganii was assessed using plasmid pT7-3 luxCDABE. No significant differences in growth and histamine formation characteristics of the lux -marked strains and wild type M. morganii strain were observed. Luminescent strain of M. morganii was used in experiments in which the correlation between light output, viable cell count and histamine formation was assessed. During the exponential growth phase, a positive linear correlation was observed between these three parameters in trypticase soy broth-histidine medium at 37C. It was demonstrated that expression of bioluminescence had not had a significant effect upon both growth rate and histamine production. Thus, the measurement of bioluminescence was found to be a simple, fast and reliable method for determination of viable cell count and histamine content. PRACTICAL APPLICATIONS Constructing predictive models in microbiology requires a large number of data on desired factors. Commonly used traditional methods of counting viable cells and measuring histamine, e.g., to model the growth limits of M. morganii as a function of different intrinsic and extrinsic factors, are time consuming and laborious, and require a lot of laboratory space and materials. According to the results of this research, measurement of bioluminescence is a simple, fast and reliable method for the determination of viable cell count and histamine content during the exponential growth phase. Thus, it can be used as a labor- and material-saving selective data capture method for constructing predictive models in many different areas. [source] Differential Glucose and Fructose Utilization During Cucumber Juice FermentationJOURNAL OF FOOD SCIENCE, Issue 1 2001Z. Lu ABSTRACT: Glucose (32 mM) and fructose (36mM) of cucumber juice were degraded simultaneously by Lactobacillus plantarum, but at different rates and extents. Glucose depletion was slightly more rapid than fructose during the exponential growth phase, but slower thereafter and stopped before exhaustion. In contrast, fructose degradation continued until all naturally present fructose was exhausted. When cucumber juice was supplemented with fructose and/or glucose, the starter culture continued to ferment fructose, but not glucose, resulting in an increase in lactic acid production and a decrease in terminal pH. Fructose utilization was not affected by the presence of glucose, but the presence of fructose reduced glucose utilization. [source] Quantitative mouse model of implant-associated osteomyelitis and the kinetics of microbial growth, osteolysis, and humoral immunity,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2008Dan Li Abstract Although osteomyelitis (OM) remains a serious problem in orthopedics, progress has been limited by the absence of an in vivo model that can quantify the bacterial load, metabolic activity of the bacteria over time, immunity, and osteolysis. To overcome these obstacles, we developed a murine model of implant-associated OM in which a stainless steel pin is coated with Staphylococcus aureus and implanted transcortically through the tibial metaphysis. X-ray and micro-CT demonstrated concomitant osteolysis and reactive bone formation, which was evident by day 7. Histology confirmed all the hallmarks of implant-associated OM, namely: osteolysis, sequestrum formation, and involucrum of Gram-positive bacteria inside a biofilm within necrotic bone. Serology revealed that mice mount a protective humoral response that commences with an IgM response after 1 week, and converts to a specific IgG2b response against specific S. aureus proteins by day 11 postinfection. Real-time quantitative PCR (RTQ-PCR) for the S. aureus specific nuc gene determined that the peak bacterial load occurs 11 days postinfection. This coincidence of decreasing bacterial load with the generation of specific antibodies is suggestive of protective humoral immunity. Longitudinal in vivo bioluminescent imaging (BLI) of luxA-E transformed S. aureus (Xen29) combined with nuc RTQ-PCR demonstrated the exponential growth phase of the bacteria immediately following infection that peaks on day 4, and is followed by the biofilm growth phase at a significantly lower metabolic rate (p,<,0.05). Collectively, these studies demonstrate the first quantitative model of implant-associated OM that defines the kinetics of microbial growth, osteolysis, and humoral immunity following infection. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J. Orthop Res 26:96,105, 2008 [source] ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM THE MARINE MICROALGA NANNOCHLOROPSIS OCULATA (EUSTIGMATOPHYCEAE),JOURNAL OF PHYCOLOGY, Issue 1 2008Juan Shi Nannochloropsis oculata (Droop) D. J. Hibberd (Eustigmatophyceae), a marine eukaryotic unicellular alga, is widely used in mariculture as live feed. It is considered to be of high nutritional value owing to its high content of proteins; polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA, C20:5n3); and diverse pigments. Previous studies of this microalga focused on its taxonomy, culture, and biochemistry, but little is known at the molecular level. Establishing a molecular base is vital to understand the biological processes of this alga. Therefore, we constructed a cDNA library using algal cells grown at exponential growth phase and carried out expressed sequence tag (EST) analysis. A total of 1,960 nonredundant sequences (NRSs) were generated for N. oculata clone CS-179. Only 32.5% of NRSs showed significant similarity (E < 1e-04) to proteins registered in the GenBank nonredundant protein database. The KOG (clusters of euKaryotic Orthologous Groups) profile database returned significant hits for 490 NRSs. Analysis revealed that a large proportion of NRSs could be unique to this microalga. [source] PRODUCTION OF PARALYTIC SHELLFISH TOXINS BY APHANIZOMENON SP.JOURNAL OF PHYCOLOGY, Issue 4 2002LMECYA 31 (CYANOBACTERIA) We examined intracellular and extracellular paralytic shellfish toxins (PST) in a strain of Aphanizomenon sp. (LMECYA31) isolated from a Portuguese freshwater reservoir throughout the growth cycle and under different conditions affected by temperature and nitrate and phosphate availability. PST concentrations and compositions were greatly influenced by cell density, growth stage, and temperature and nutrients conditions. On a per-cell basis results showed (1) the enhancement of PST cell quota after the end of exponential growth phase in nutrient replete batch cultures, (2) the absence of a PST increment at late growth stages under phosphate limitation, (3) a rise in PST maximum cell quota under nitrate depletion, and (4) the enhancement of toxin production at higher temperatures. The relative proportion of the four toxins detected, neoSTX, dcSTX, STX and GTX5, also changed within and between culture settings. While growing under phosphate rich media cells produced mainly GTX5 and neoSTX, whereas under phosphate limitation the proportion of STX and dcSTX increased substantially with culture age. Large amounts of extracellular toxins were found in the culture medium, increasing during culture time. Extracellular toxin composition in each culture was fairly constant and always similar to the intracellular composition found at late stages of growth. This further supported other research that indicates that PSTs are released to the water through cell lysis, and a significant concentration of PST may be expected to remain in the water upon the collapse of a toxic bloom or after cells removal by water treatment. [source] EFFECTS OF BARLEY STRAW EXTRACT ON GROWTH OF FIVE SPECIES OF PLANKTONIC ALGAEJOURNAL OF PHYCOLOGY, Issue 2001Article first published online: 24 SEP 200 Holz, J. C.1, Fessler, C. J.2, Severn, A. A.1 & Hoagland, K. D.1 1School of Natural Resource Sciences, University of Nebraska, 103 Plant Industry Bldg., Lincoln, NE, 68583-0814; 2Biology Department, Nebraska Wesleyan University, 5000 St. Paul Ave., Lincoln, NE, 68504; Phone: 402-472-6648; Fax: 402-472-2964 The effects of exposure to barley straw extract and the timing of exposure on the growth of four common cyanophyte species and one species of green algae were investigated in two laboratory experiments. Clonal cultures of Anabaena cylindrica, Cylindrospermum sp., Gloeocapsa sp., Eucapsis sp., and Chlorella vulgaris were obtained from culture collections. In both experiments, the algae were cultured in Guillard's WC medium at 20 °C on a 12:12 L/D photoperiod. In the first experiment, the algae were dosed with four concentrations of barley straw extract at the beginning of the experiment (day 0) and growth was monitored every second day using fluorometric detection of chlorophyll a for 14 d. In the second experiment, the algae were dosed with the same extract concentrations, but the extract was not added until the algae were in exponential growth phase (day 6). Both experiments also had control treatments (i.e. no extract) and each extract and control treatment was replicated five times. Growth of C. vulgaris was inhibited by all doses in both experiments, but inhibition was 22% greater when the extract was added on day 0. Growth Gleocapsa sp. was slightly inhibited by all doses when the extract was added on day 0, but not when it was added on day 6. No other species were inhibited, regardless of dose or timing of dose. The results of this study and other bioassay studies suggest that differential susceptibility to barley straw among algae is common and may reduce the effectiveness of barley straw as an algal control technique. [source] Hypersalinity enhances the production of extracellular polymeric substance (eps) in the texas brown tide alga, aureoumbra lagunensis (PELAGOPHYCEAE)JOURNAL OF PHYCOLOGY, Issue 1 2000Hongbin Liu Laboratory experiments with batch cultures showed that the Texas brown tide alga, Aureoumbra lagunensis Stockwell, DeYoe, Hargraves et Johnson, produced a large amount of extracellular polymeric substance (EPS) that formed an extracellular polysaccharide mucus layer. Both dissolved and particulate carbohydrate and EPS concentrations increased as A. lagunensis cultures progressed from exponential growth phase, through stationary phase, to declining phase. Particulate carbohydrate and EPS concentrations per cell were more than three times higher during the declining phase than that in exponential growth phase, reflecting a large increase in the EPS mucus layer. The amounts of carbohydrate and EPS produced by A. lagunensis were significantly higher under hypersaline conditions. The thicker EPS mucus layer surrounding A. lagunensis cells under hypersaline conditions might be a protective adaptation that permits it to bloom under hypersaline conditions that most other phytoplankton cannot survive. This could be one of the reasons why the Texas brown tide persisted in the Laguna Madre, an often hypersaline coastal lagoon, for 7 years. [source] Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilmsMOLECULAR MICROBIOLOGY, Issue 1 2004Irene Kuiper Summary Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High-performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface-tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface-active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N-terminus of a 12-amino-acid peptide moiety, in which the C-terminal carboxylic acid group forms an ester with the hydroxyl side-chain of Ser9. The difference between the two structures is located in the second amino acid from the C-terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio-synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild-type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms. [source] The CitST two-component system regulates the expression of the Mg-citrate transporter in Bacillus subtilisMOLECULAR MICROBIOLOGY, Issue 4 2000Hiroki Yamamoto citS and citT genes encoding a new two-component system were identified in the 71° region between the pel and citM loci on the Bacillus subtilis chromosome. citS- and citT- deficient strains were unable to grow on minimal plates including citrate as a sole carbon source. In addition, a strain deficient in citM, which encodes the secondary transporter of the Mg-citrate complex, exhibited the same phenotype on this medium. Northern blot analysis revealed that citM was polycistronically transcribed with the downstream yflN gene, and that CitS and CitT were necessary for transcription of the citM,yflN operon. Upon addition of 2 mM citrate to DSM, this operon was strongly induced after the middle of the exponential growth phase in the wild type, but not in the citST double null mutant. Moreover, the transcription of this operon was completely repressed in the presence of 1% glucose. We found a sequence exhibiting homology to a catabolite-responsive element (cre) in the citM promoter region. Glucose repression was lost in ccpA and citM,cre mutants. From the result of a citM,promoter deletion experiment, putative CitT target sequences were found to be located around two regions, from ,62 to ,74 and from ,149 to ,189, relative to the citM start point. Furthermore, DNase I footprinting assays revealed that these two CitT target regions extended maximally from ,36 to ,84 and from ,168 to ,194. From these findings, we concluded that the expression of citM is positively regulated by the CitST system and negatively regulated by CcpA. [source] Growth Phase and Elemental Stoichiometry of Bacterial Prey Influences Ciliate Grazing SelectivityTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 5 2009DAVID F. GRUBER ABSTRACT. Protozoa are known to selectively graze bacteria and can differentiate prey based on size and viability, but less is known about the effects of prey cellular composition on predator selectivity. We measured the effect of growth phase and elemental stoichiometry of Escherichia coli on grazing by two ciliates, Euplotes vannus and Cyclidium glaucoma. Bacterial cells of a single strain were transformed with green and red fluorescent protein and harvested from culture at differing growth stages. Cells in exponential growth phase had low carbon:phosphorus (39) and nitrogen:phosphorus (9) ratios, while cells from stationary phase had high carbon:phosphorus of 104 and nitrogen:phosphorus of 26. When offered an equal mixture of both types of bacteria, Cyclidium grazed stationary phase, high carbon:phosphorus, high nitrogen:phosphorus cells to 22% of initial abundance within 135 min, while Euplotes reduced these cells to 33%. Neither ciliate species decreased the abundance of the exponential phase cells, lower carbon:phosphorus and nitrogen:phosphorus, relative to control treatments. Because protozoa have higher nitrogen:phosphorus and carbon:phosphorus ratios than their prokaryotic prey, this study raises the possibility that it may be advantageous for protozoa to preferentially consume more slowly growing bacteria. [source] Proliferation and apoptosis in a neuroblastoma cell line exposed to 900 MHz modulated radiofrequency fieldBIOELECTROMAGNETICS, Issue 3 2006P. Merola Abstract The aim of this study was to examine whether a modulated radiofrequency of the type used in cellular phone communications at a specific absorption rate (SAR) higher than International Commission on Non-ionizing Radiation Protection (ICNIRP) reference level for occupational exposure, could elicit alterations on proliferation, differentiation, and apoptosis processes in a neuroblastoma cell line. The cell line was exposed for 24, 48, and 72 h to 900 MHz radiofrequency and proliferation and differentiation were tested by WST-I assay and by a molecular analysis of specific markers, two oncogenes and a cytoskeleton protein, in exponential growth phase and in synchronized cell cultures. Apoptosis was evaluated by caspase activation analysis and by molecular detection of Poly (ADP-ribose) polimerase (PARP) cleavage. Combined exposures to radiofrequency and to the differentiative agent retinoic acid or to the apoptotic inducer camptothecin were carried out to test possible interference between electromagnetic field and chemical agents. Overall our data suggest that 900 MHz radiofrequency exposure up to 72 h does not induce significant alterations in the three principal cell activities in a neuroblastoma cell line. Bioelectromagnetics 27:164,171, 2006. © 2006 Wiley-Liss, Inc. [source] Twenty-four-well plate miniature bioreactor high-throughput system: Assessment for microbial cultivationsBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2007Kevin Isett Abstract High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (µ)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the µ-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6,±,2.4, 46.5,±,4.6, 51.6,±,3.7, and 56.1,±,1.6 h,1 at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively. Biotechnol. Bioeng. 2007;98: 1017,1028. © 2007 Wiley Periodicals, Inc. [source] Optimization of fed-batch parameters and harvest time of CHO cell cultures for a glycosylated product with multiple mechanisms of inactivationBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2007Ryan S. Senger Abstract Optimization of fed-batch feeding parameters was explored for a system with multiple mechanisms of product inactivation. In particular, two separate mechanisms of inactivation were identified for the recombinant tissue-type activator (r-tPA) protein. Dynamic inactivation models were written to describe particular r-tPA glycoform inactivation in the presence and absence of free-glucose. A glucose-independent inactivation mechanism was identified, and inactivation rate constants were found dependent upon the presence of glycosylation of r-tPA at N184. Inactivation rate constants of the glucose-dependent mechanism were not affected by glycosylation at N184. Fed-batch optimization was performed for r-tPA production by CHO cell culture in a stirred-tank reactor with glucose, glutamine and asparagine feed. Feeding profiles in which culture supernatant concentrations of free-glucose and amino acids (combined glutamine and asparagine) were used as control variables, were evaluated for a wide variety of set points. Simulation results for a controlled feeding strategy yielded an optimum at set points of 1.51 g L,1 glucose and 1.18 g L,1 of amino acids. Optimization was also performed in absence of metabolite control using fixed feed-flow rates initiate during the exponential growth phase. Fixed feed-flow results displayed a family of optimum solutions along a mass flow rate ratio of 3.15 of glucose to amino acids. Comparison of the two feeding strategies showed a slight advantage of rapid feeding at a fixed flow rate as opposed to metabolite control for a product with multiple mechanisms of inactivation. Biotechnol. Bioeng. 2007;98: 378,390. © 2007 Wiley Periodicals, Inc. [source] Study of stationary phase metabolism via isotopomer analysis of amino acids from an isolated proteinBIOTECHNOLOGY PROGRESS, Issue 1 2010Afshan S. Shaikh Abstract Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully 13C-labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Good modeling practice for PAT applications: Propagation of input uncertainty and sensitivity analysisBIOTECHNOLOGY PROGRESS, Issue 4 2009Gürkan Sin Abstract The uncertainty and sensitivity analysis are evaluated for their usefulness as part of the model-building within Process Analytical Technology applications. A mechanistic model describing a batch cultivation of Streptomyces coelicolor for antibiotic production was used as case study. The input uncertainty resulting from assumptions of the model was propagated using the Monte Carlo procedure to estimate the output uncertainty. The results showed that significant uncertainty exists in the model outputs. Moreover the uncertainty in the biomass, glucose, ammonium and base-consumption were found low compared to the large uncertainty observed in the antibiotic and off-gas CO2 predictions. The output uncertainty was observed to be lower during the exponential growth phase, while higher in the stationary and death phases - meaning the model describes some periods better than others. To understand which input parameters are responsible for the output uncertainty, three sensitivity methods (Standardized Regression Coefficients, Morris and differential analysis) were evaluated and compared. The results from these methods were mostly in agreement with each other and revealed that only few parameters (about 10) out of a total 56 were mainly responsible for the output uncertainty. Among these significant parameters, one finds parameters related to fermentation characteristics such as biomass metabolism, chemical equilibria and mass-transfer. Overall the uncertainty and sensitivity analysis are found promising for helping to build reliable mechanistic models and to interpret the model outputs properly. These tools make part of good modeling practice, which can contribute to successful PAT applications for increased process understanding, operation and control purposes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] A Procedure for High-Yield Spore Production by Bacillus s ubtilisBIOTECHNOLOGY PROGRESS, Issue 4 2005Sandra M. Monteiro Bacillus subtilis spores have a number of potential applications, which include their use as probiotics and competitive exclusion agents to control zoonotic pathogens in animal production. The effect of cultivation conditions on Bacillus subtilis growth and sporulation was investigated in batch bioreactions performed at a 2-L scale. Studies of the cultivation conditions (pH, dissolved oxygen concentration, and media composition) led to an increase of the maximum concentration of vegetative cell from 2.6 × 109 to 2.2 × 1010 cells mL - 1 and the spore concentration from 4.2 × 108 to 5.6 × 109 spores mL - 1. A fed-batch bioprocess was developed with the addition of a nutrient feeding solution using an exponential feeding profile obtained from the mass balance equations. Using the developed feeding profile, starting at the middle of the exponential growth phase and finishing in the late exponential phase, an increase of the maximum vegetative cell concentration and spore concentration up to 3.6 × 1010 cells mL - 1 and 7.4 × 109 spores mL - 1, respectively, was obtained. Using the developed fed-batch bioreaction a 14-fold increase in the concentration of the vegetative cells was achieved. Moreover, the efficiency of sporulation under fed-batch bioreaction was 21%, which permitted a 19-fold increase in the final spore concentration, to a final value of 7.4 × 109 spores mL - 1. This represents a 3-fold increase relative to the highest reported value for Bacillus subtilis spore production. [source] Insight Into the Kinetic of Amyloid , (1,42) Peptide Self-Aggregation: Elucidation of Inhibitors' Mechanism of ActionCHEMBIOCHEM, Issue 17 2007Manuela Bartolini Dr. Abstract The initial transition of amyloid , (1,42) (A,42) soluble monomers/small oligomers from unordered/,-helix to a ,-sheet-rich conformation represents a suitable target to design new potent inhibitors and to obtain effective therapeutics for Alzheimer's disease. Under optimized conditions, this reliable and reproducible CD kinetic study showed a three-step sigmoid profile that was characterized by a lag phase (prevailing unordered/,-helix conformation), an exponential growth phase (increasing ,-sheet secondary structure) and a plateau phase (prevailing ,-sheet secondary structure). This kinetic analysis brought insight into the inhibitors' mechanism of action. In fact, an increase in the duration of the lag phase can be related to the formation of an inhibitor,A, complex, in which the non-amyloidogenic conformation is stabilized. When the exponential rate is affected exclusively, such as in the case of Congo red and tetracycline, then the inhibitor affinity might be higher for the pleated ,-sheet structure. Finally, by adding the inhibitor at the end of the exponential phase, the soluble protofibrils can be disrupted and the A, amyloidogenic structure can revert into monomers/small oligomers. Congo red and tetracycline preferentially bind to amyloid in the ,-sheet conformation because both decreased the slope of the exponential growth, even if to a different extent, whereas no effect was observed for tacrine and galantamine. Some very preliminary indications can be derived about the structural requirements for binding to nonamyloidogenic or ,-sheet amyloid secondary structure for the development of potent antiaggregating agents. On these premises, memoquin, a multifunctional molecule that was designed to become a drug candidate for the treatment of Alzheimer's disease, was investigated under the reported circular dichroism assay and its anti-amyloidogenic mechanism of action was elucidated. [source] | ||||||||||||||||