Myceloid cell formation in Arthrobacter globiformis during osmotic stress

C.E. DEUTCH AND G.S. PERERA. 1992. Arthrobacter globiformis was grown in a semi-defined liquid medium containing added solutes to determine the effects of osmotic stress on its reproduction and cell morphology. There was a progressive reduction in the specific growth rate during exponential phase as the concentration of NaCl was increased, although the final yields of the cultures during stationary phase were not affected. Clusters of branching myceloid cells rather than the typical bacillary forms predominated during exponential phase. These myceloids did not undergo complete septation and persisted into stationary phase. Similar responses were observed with potassium sulphate as the exogenous solute but less dramatic morphological effects were found with added polyethylene glycol or sucrose. The myceloids formed in response to osmotic stress could not be disrupted mechanically but were more sensitive than normal cells to lysozyme, particularly during stationary phase. Addition of osmoprotective compounds such as proline, glutamate, glycine betaine, or trehalose to the growth medium did not significantly relieve the effects of osmotic stress on growth rate or morphology. A. simplex also formed myceloid cells during osmotic stress but A. crystallopoietes did not. These results indicate that arthrobacters exhibit characteristic responses to osmotic stress and suggest these bacteria may contain novel osmoprotective compounds.     

Myceloid cell formation in Arthrobacter globiformis during osmotic stress.

Arthrobacter globiformis was grown in a semi-defined liquid medium containing added solutes to determine the effects of osmotic stress on its reproduction and cell morphology. There was a progressive reduction in the specific growth rate during exponential phase as the concentration of NaCl was increased, although the final yields of the cultures during stationary phase were not affected. Clusters of branching myceloid cells rather than the typical bacillary forms predominated during exponential phase. These myceloids did not undergo complete septation and persisted into stationary phase. Similar responses were observed with potassium sulphate as the exogenous solute but less dramatic morphological effects were found with added polyethylene glycol or sucrose. The myceloids formed in response to osmotic stress could not be disrupted mechanically but were more sensitive than normal cells to lysozyme, particularly during stationary phase. Addition of osmoprotective compounds such as proline, glutamate, glycine betaine, or trehalose to the growth medium did not significantly relieve the effects of osmotic stress on growth rate or morphology. A. simplex also formed myceloid cells during osmotic stress but A. crystallopoietes did not. These results indicate that arthrobacters exhibit characteristic responses to osmotic stress and suggest these bacteria may contain novel osmoprotective compounds.     

Physiological and morphological changes during short term starvation of marine bacterial islates

Three marine bacteria were examined for physiological and morphological changes in the initial phase of starvation. It was found that the starvation process was induced in a similar way irrespective of whether the cells were suspended in nutrient and energy free artificial seawater (NSS) or NSS supplemented with nitrogen and phosphorus. An initial phase of increased activity was consistent with a decreased response to added nutrients. Recovery from starvation exhibited the same response in both these starvation regimes, measured throughout the starvation period. Cells in nitrogen or phosphorus deprived starvation regimes, showed a high and rapid increased activity, followed by a delayed and more pronounced decline in respiratory activity. The initial phase of starvation also included a loss of poly--hydroybutyrate as observed by transmission electron microscopy (TEM). Two bacterial strains showed formation of small vesicles on the outer cell layer when examined by TEM. This formation and release of vesicles was related to the continuous size reduction during starvation survival. The results are discussed in terms of defining the mechanisms of initial cellular responses to nutrient deprivation.Abbreviation NSS nine salt solution     

Growth of <Emphasis Type="Italic">Paenarthrobacter aurescens</Emphasis> strain TC1 on atrazine and isopropylamine during osmotic stress

Paenarthrobacter aurescens strain TC1 can use the herbicide atrazine and its degradation product isopropylamine as nutrients. Because osmotic stress can change the morphology of arthrobacters and decrease their metabolism of some carbon compounds, the effects of increasing NaCl concentrations on strain TC1 and its ability to utilize atrazine and isopropylamine were determined. Strain TC1 was cultured in minimal media with different NaCl concentrations and varying combinations of d-glucose, ammonium sulfate, atrazine, or isopropylamine. Growth was measured quantitatively as an increase in turbidity. Physiological effects were assessed using Biolog? GP test plates and BD BBL Crystal GP or bioMérieux API 20E test systems. The effects of osmoprotective compounds were determined in liquid media and on agar plates. Strain TC1 formed multicellular myceloids and its growth rate slowed as the salt concentration increased, but the culture yields were similar up to 0.6 mol l^?1 NaCl. The bacteria metabolized about half the carbon sources in Biolog? GP test plates, but their use of some compounds and several hydrolytic activities decreased with high salt concentrations. However, strain TC1 grew well with atrazine and isopropylamine as the nitrogen source in media containing up to 0.6 mol l^?1 NaCl. Growth in 0.8 mol l^?1 NaCl was more limited but could be enhanced by glycine betaine, L-proline, and L-glutamate. P. aurescens strain TC1 can continue to use atrazine and isopropylamine as nutrients during osmotic stress and so may be particularly useful for remediation of contaminated soils with low water activity.     

Structural and physiological diversity among cystlike resting cells of bacteria of the genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

Cystlike resting cells (CRC) of non-spore-forming gram-negative bacteria of the genus Pseudomonas, P. aurantiaca and P. fluorescens, were obtained and characterized for the first time; their physiological and morphological diversity was demonstrated. The following properties were common for all the revealed types of CRC as dormant forms: (1) long-term (up to 6 months or longer) maintenance of viability in the absence of culture growth and cell respiration; (2) absence of an experimentally detectable level of metabolism; (3) higher resistance to damage and autolysis under the action of provoking factors than in metabolically active vegetative cells; and (4) specific features of ultrastructural organization absent in vegetative cells: thickened and lamellar envelopes, clumpy structure of the cytoplasm, and condensed DNA in nucleoid. The differences in various types of CRC concern the thickness and lamellar structure of cell envelopes, as well as the presence and thickness of the capsular layer. In particular, forms ultrastructurally similar to typical bacterial cysts were revealed in pseudomonad populations growing on soil agar. Physiological diversity was revealed in different levels of viability preservation and thermal resistance in various types of CRC and depended on the conditions of their formation. The optimal conditions and procedures for obtaining P. aurantiaca and P. fluorescens CRC that retain the ability to form colonies on standard nutrient media are as follows: (1) a twofold decrease of nitrogen content in the growth medium; (2) an increased level of anabiosis autoinducer (C₁₂-AHB, 10^?4 M) in stationary cultures; (3) transfer of the cells from stationary cultures to a starvation medium with silica; (4) cultivation in soil extract; and (5) development of cultures on soil agar. The CRC from the cultures grown in soil extract or starvation medium with silica proved to be resistant to heat treatment (60°C, 5 min). In the CRC formed in nitrogen-limited media, the degree of heat resistance increased at longer incubation (1.5 to 6 months). CRCs on soil agar surface were resistant to desiccation. The ultrastructure of the morphologically varied types of P. aurantiaca CRC formed under simulated natural conditions is described for the first time. The data on the intraspecies diversity of pseudomonad dormant forms contribute to the concept of plasticity of the life style and adaptive reactions that ensure survival of these bacteria in unfavorable environmental conditions.     

Nutritional control of copper uptake in the cyanobacteriumNostoc calcicola Bréb

Summary The kinetics of Cu uptake in nutritionally starved cells of the diazotrophic cyanobacteriumNostoc calcicola Bréb. have been compared with those in cells recovering from starvation. Unstarved cyanobacterial cells assimilated 97.0 nmol Cu mg^–1 protein within 1 h when incubated in medium containing 40 M Cu. Uptake was markedly inhibited in carbon-starved cells and, to a lesser extent, in cells starved of nitrogen or sulphur. The intracellular concentrations of protein and photopigments were markedly lower in cells starved of carbon, nitrogen, sulphur or phosphorus, whilst that of carbohydrate was lower in cells starved of carbon, sulphur or phosphorus, but almost doubled in cells starved of nitrogen. The ability to assimilate Cu was partially restored in cells after 72 h of recovery from phosphorus or sulphur deprivation, but showed little improvement during recovery from carbon or nitrogen starvation. A possible role of phosphorus in regulating Cu transport and accumulation is discussed.     

Asparagine metabolism and nitrogen distribution during protein degradation in sugar-starved maize root tips

Excised maize (Zea mays L.) root tips were used to monitor the effects of prolonged glucose starvation on nitrogen metabolism. Following root-tip excision, sugar content was rapidly exhausted, and protein content declined to 40 and 8% of its initial value after 96 and 192 h, respectively. During starvation the contents of free amino acids changed. Amino acids that belonged to the same synthetic family showed a similar pattern of changes, indicating that their content, during starvation, is controlled mainly at the level of their common biosynthetic steps. Asparagine, which is a good marker of protein and amino-acid degradation under stress conditions, accumulated considerably until 45 h of starvation and accounted for 50% of the nitrogen released by protein degradation at that time. After 45 h of starvation, nitrogen ceased to be stored in asparagine and was excreted from the cell, first as ammonia until 90–100 h and then, when starvation had become irreversible, as amino acids and aminated compounds. The study of asparagine metabolism and nitrogen-assimilation pathways throughout starvation showed that: (i) asparagine synthesis occurred via asparagine synthetase (EC 6.3.1.1) rather than asparagine aminotransferase (EC 2.6.1.14) or the -cyanoalanine pathway, and asparagine degradation occurred via asparaginase (EC 3.5.1.1); and (ii) the enzymic activities related to nitrogen reduction and assimilation and amino-acid synthesis decreased continuously, whereas glutamate dehydrogenase (EC 1.4.1.2–4) activities increased during the reversible period of starvation. Considered together, metabolite analysis and enzymic-activity measurements showed that starvation may be divided into three phases: (i) the acclimation phase (0 to 30–35 h) in which the root tips adapt to transient sugar deprivation and partly store the nitrogen released by protein degradation, (ii) the survival phase (30–35 to 90–100 h) in which the root tips expel the nitrogen released by protein degradation and starvation may be reversed by sugar addition and (iii) the cell-disorganization phase (beyond 100 h) in which all metabolites and enzymic activities decrease and the root tips die.Abbreviations AlaAT alanine aminotransferase - AspAT aspartate aminotransferase - AS asparagine synthetase - Asnase asparaginase - AsnAT asparagine aminotransferase - -CS -cyanoalanine synthase - GDH glutamate dehydrogenase - Glnase glutaminase - GOGAT glutamate synthase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

Changes in body composition of germ-free and conventional chickens during starvation

• 1.1. Changes in body composition during starvation were compared between germ-free (GF) and conventionalized (CVL) chicks in experiment 1. At 8 days of age, the GF birds were divided into two groups, i.e. GF and CVL groups. The CVL birds were inoculated with faeces from conventionally reared birds. Until 14 days of age, both birds were fed a diet ad lib, and thereafter starved for 6 days.
• 2.2. Nitrogen loss during starvation was significantly lower in CVL birds, though the reverse was true for water loss. Fasting heat production was comparable between two environments.
• 3.3. Influence of the gut microflora on body weight and nitrogen losses during starvation was investigated in birds prefed diets high or low in dietary protein in experiment 2.
• 4.4. No significant effect of the gut microflora was observed in body weight and nitrogen losses. Body weight was severely reduced in birds prefed the high protein diet and nitrogen loss was lower in birds prefed the low protein diet.

     

Growth responses of blue-green algae to sodium chloride concentration

Summary General characteristics of blue-green algal halotolerance were studied by growth experiments and selected analyses. Variation in NaCl concentration was used to mimic salinity. Marine isolates were more halotolerant (8–10% NaCl) than non-marine isolates (2% NaCl). The Na⁺ requirement for growth was saturated at 1 mg NaCl/l for non-marine isolates and 100mg NaCl/l for marine isolates. Intracellular Na⁺ values were affected by washing; however, bound-K⁺ values for both marine and fresh-water blue-green algae were fairly constant, 1–3 g/mg cells. A specific Na⁺ function was implied by the retention after washing of ²²Na⁺ (0.1 g/mg cells) by Agmenellum quadruplicatum (PR-6), a marine coccoid blue-green alga.High concentrations of NaCl apparently inhibit growth more by ionic (Na⁺) stress than by osmotic stress. Changes in light, temperature, pH, or composition of the basal medium failed to alleviate this stress.In contrast to marine bacteria, cells of PR-6 grown in Medium ASP-2+90 g NaCl/l did not undergo lysis when suspended in distilled water. However, viability of cells grown in Medium ASP-2+90 g NaCl/l decreased rapidly compared to cells grown in Medium ASP-2+18 g NaCl/l.Cells of PR-6 grown in ASP-2+90 g NaCl/l were larger than normal, formed chains (3–16 cells), and appeared bleached. Analyses of such cells revealed an overall decrease in fatty acids, hydrocarbons, and pigment levels. Electron micrographs showed that NaCl stressed cells were little altered in morphology.The photosynthesis of PR-6 cells was immediately depressed when the cells were transferred from 18 g NaCl/l to 70 g NaCl/l medium. When held in the latter for several hours the rate recovered and approached the initial photosynthetic rate maintained before NaCl-shock. This phenomenon was never seen with non-marine isolates. The explanation may lie in the ability of the cell to adjust to sudden Na⁺ increase via an ion (Na⁺) pump, for example, adenosine triphosphatase (ATPase). Subsequent assays suggested more ATPase activity in a marine isolated than in a nonmarine isolate. The ATPase was not, however, ouabain sensitive.It is suggested that marine blue-green algal isolates are characteristically more halotolerant, perhaps by selection, than fresh-water forms. This difference may be due in part to inherent capacity of the cell to extrude Na⁺. Alternatively, in freshwater forms rhe Na⁺ functional sites may be more Na⁺ sensitive than in marine forms.     

Correlation between NaCl Sensitivity of Rhizobium Bacteria and Ineffective Nodulation of Leguminous Plants

A sodium chloride (NaCl)-sensitive mutant of Rhizobium fredii USDA191, which contained a single copy of Tn5-Mob transposed into chromosomal DNA, was obtained by Tn5-Mob random insertion. The growth rate of this mutant was lower than that of the wild type in the presence of 0.2 M NaCl and it seemed to lack the inductive ATP production in response to the addition of NaCl. This mutant induced the formation of small and whitish nodules on lateral roots of soybeans, which were negative for acetylene reduction activity, indicating that the nodules were ineffective for nitrogen fixation. The mutant also reduced the weight of above-ground portions and roots to 64 and 55%, respectively, compared with the weight of the plants inoculated with the wild-type cells. These results suggest that NaCl sensitivity of Rhizobium bacteria is one of the important factors for nodule formation and nitrogen fixation.     

Comparative proteomic analysis between nitrogen supplemented and starved conditions in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Background

Fungi are constantly exposed to nitrogen limiting environments, and thus the efficient regulation of nitrogen metabolism is essential for their survival, growth, development and pathogenicity. To understand how the rice blast pathogen Magnaporthe oryzae copes with limited nitrogen availability, a global proteome analysis under nitrogen supplemented and nitrogen starved conditions was completed.

Methods

M. oryzae strain 70–15 was cultivated in liquid minimal media and transferred to media with nitrate or without a nitrogen source. Proteins were isolated and subjected to unfractionated gel-free based liquid chromatography-tandem mass spectrometry (LC-MS/MS). The subcellular localization and function of the identified proteins were predicted using bioinformatics tools.

Results

A total of 5498 M. oryzae proteins were identified. Comparative analysis of protein expression showed 363 proteins and 266 proteins significantly induced or uniquely expressed under nitrogen starved or nitrogen supplemented conditions, respectively. A functional analysis of differentially expressed proteins revealed that during nitrogen starvation nitrogen catabolite repression, melanin biosynthesis, protein degradation and protein translation pathways underwent extensive alterations. In addition, nitrogen starvation induced accumulation of various extracellular proteins including small extracellular proteins consistent with observations of a link between nitrogen starvation and the development of pathogenicity in M. oryzae.

Conclusion

The results from this study provide a comprehensive understanding of fungal responses to nitrogen availability.

     

Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold

Genetically engineered rice (Oryza sativa L.) with the ability to synthesize glycinebetaine was established by introducing the codA gene for choline oxidase from the soil bacterium Arthrobacter globiformis. Levels of glycinebetaine were as high as 1 and 5 mol per gram fresh weight of leaves in two types of transgenic plant in which choline oxidase was targeted to the chloroplasts (ChlCOD plants) and to the cytosol (CytCOD plants), respectively. Although treatment with 0.15 m NaCl inhibited the growth of both wild-type and transgenic plants, the transgenic plants began to grow again at the normal rate after a significantly less time than the wild-type plants after elimination of the salt stress. Inactivation of photosynthesis, used as a measure of cellular damage, indicated that ChlCOD plants were more tolerant than CytCOD plants to photoinhibition under salt stress and low-temperature stress. These results indicated that the subcellular compartmentalization of the biosynthesis of glycinebetaine was a critical element in the efficient enhancement of tolerance to stress in the engineered plants.     

Requirement of the isocitrate lyase gene <Emphasis Type="Italic">ICL1</Emphasis> for <Emphasis Type="Italic">VPS41</Emphasis>-mediated starvation response in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans is a major cause of fungal meningitis in individuals with impaired immunity. Our previous studies have shown that the VPS41 gene plays a critical role in the survival of Cryptococcus neoformans under nitrogen starvation; however, the molecular mechanisms underlying VPS41-mediated starvation response remain to be elucidated. In the present study, we show that, under nitrogen starvation, VPS41 strongly enhanced ICL1 expression in C. neoformans and that overexpression of ICL1 in the vps41 mutant dramatically suppressed its defects in starvation response due to the loss of VPS41 function. Moreover, targeted deletion of ICL1 resulted in a dramatic decline in viability of C. neoformans cells under nitrogen deprivation. Taken together, our data suggest a model in which VPS41 up-regulates ICL1 expression, directly or indirectly, to promote survival of C. neoformans under nitrogen starvation.     

Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients.

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.     

Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients.

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.     

Synthesis of unique proteins at the onset of carbon starvation inEscherichia coli

Summary Escherichia coli bulk protein synthesis continued during the first 3–4 h of carbon starvation at 50–75% that of non-starved (growing) cells. Two-dimensional gel electrophoresis analysis of in vivo pulse-labelled proteins resolved at least 30 polypeptides with new or increased synthesis, relative to total protein synthesis, during this time. Among these polypeptides were several that were also synthesized by ethanol-treatedE. coli (heat-shock proteins). In addition, a number of unique polypeptides were synthesized by carbon-starved cells. These starvation proteins may be involved in survival of the starving bacteria.     

Analysis of the Variability in the Number of Viable Bacteria after Mild Heat Treatment of Food

Variability in the numbers of bacteria remaining in saline solution and whole milk following mild heat treatment has been studied with Listeria innocua, Enterococcus faecalis, Salmonella enterica serovar Enteritidis, and Pseudomonas fluorescens. As expected, the most heat-resistant bacterium was E. faecalis, while P. fluorescens was the least heat resistant, and all bacteria showed greater thermal resistance in whole milk than in saline solution. Despite the differences in the inactivation kinetics of these bacteria in different media, the variability in the final number of bacteria was affected neither by the species nor by the heating substrate, but it did depend on the intensity of the heat treatment. The more severe the heat treatment was, the lower the average number of surviving bacteria but the greater the variability. Our results indicated that the inactivation times for the cells within a population are not identically distributed random variables and that, therefore, the population includes subpopulations of cells with different distributions for the heat resistance parameters. A linear relationship between the variability of the log of the final bacterial concentration and the logarithmic reduction in the size of the bacterial population was found.The safety and quality of many foods, such as minimally processed foods and some ready-to-eat products, are affected by a combination of multiple sublethal stresses due to processing and storage conditions (17, 32). The use of mild preservative processes instead of harsher ones affects the microbiological risk. It is therefore important to identify potential hazards and to perform accurate quantitative microbial risk assessments including probabilistic modeling approaches (17).It is unlikely that food products are contaminated by one unique bacterial strain or species. In fact, even cells from a pure culture derived from one cell are not necessarily identical (25). Nauta (16) differentiated between uncertainty, which is due to the lack of perfect knowledge about a parameter and which is reducible by further measurements, and variability, which reflects the true variability of a population and is therefore irreducible. He showed the importance of both parameters in quantitative microbial risk assessment models. The estimation of variability usually refers to the variation in the responses of the individual cells within the population, which may be homogeneous or heterogeneous. In a homogeneous population, the heat resistance parameters of the individual cells are identically distributed random variables. Heterogeneous populations include subpopulations of cells with different distributions for the heat resistance parameters. The heterogeneity of the individual cells or spores within a population is the most accepted explanation for the tails of inactivation curves (1, 30) and unexpected increases in the variability of the number of survivors after heat treatments (7, 8, 30).The microbial concentration in a food product at a given time is affected by the initial inoculum, the length of the lag phase, and the maximum specific growth rate. The distributions of these variables are required to estimate the distribution of the log of the bacterial concentration at a given time during exponential growth (2). Typically for these estimates, the growth rate is assumed to be constant during the exponential growth phase in a nonfluctuating environment. Under this assumption, variations in the growth rate are assumed to be caused by lack of control over the environmental conditions. Recently, distributions of the length of the lag phase among the surviving cells after different stresses have been measured (6, 9, 14, 18). Usually, the distribution of the initial number of cells in a food product is assumed to be independent of previous processing and storage conditions. However, this may be not the case following stress conditions such as heat treatments. The distribution of the initial number of bacteria may depend on the severity of the previous stress. The purpose of the present work was to analyze the distributions of the concentrations of surviving cells after different heat treatments. To do this, heat treatments of various intensities were applied to the gram-positive bacteria Enterococcus faecalis and Listeria innocua, as well as to the gram-negative bacteria Salmonella enterica serovar Enteritidis and Pseudomonas fluorescens.     

Impacts of nitrogen and phosphorus starvation on the physiology of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The importance of algae-derived biofuels has been highlighted by the current problems associated with fossil fuels. Considerable past research has shown that limiting nutrients such as nitrogen and phosphorus increases the cellular lipid content in microalgae. However, limiting the supply of nutrients results in decreased biomass, which in turn decreases the overall lipid productivity of cultures. Therefore, nutrient limitation has been a subject of dispute as to whether it will benefit biofuel production on an industrial scale. Our research explores the physiological changes a cell undergoes when exposed to nitrogen and phosphorus limitations, both individually and in combination, and also examines the biotechnological aspects of manipulating N and P in order to increase cellular lipids, by analyzing the lipid production. We show that nitrogen starvation and also nitrogen plus phosphorus starvation combined have a more profound effect on the physiology and macromolecular pools of Chlamydomonas reinhardtii than does phosphorus starvation alone. The photosynthetic performance of C. reinhardtii underwent drastic changes under nitrogen starvation, but remained relatively unaffected under phosphorus starvation. The neutral lipid concentration per cell was at least 2.4-fold higher in all the nutrient-starved groups than the nutrient-replete controls, but the protein level per cell was lower in the nitrogen-starved groups. Overall, nitrogen starvation has a more dramatic effect on the physiology and neutral lipids and protein levels of C. reinhardtii than phosphorus starvation. However, the level of total lipids per volume of culture obtained was similar among nutrient-replete and all of the nutrient-starved groups. We conclude that combined nitrogen and phosphorus starvation does not likely benefit biofuel production in terms of enhanced lipid or biomass production.     

Changes in growth,osmotic potential and cell permeability of wheat cultivars under salt stress

10-d-old wheat seedlings were grown hydroponically in presence and absence of 100 mM NaCl for 7 d. Salt stress decreased growth of shoots and roots of both cultivars; fresh mass of sensitive cultivar being more affected. NaCl increased membrane permeability to urea, methylurea and ethylurea and decreased membrane partiality in root cortex cells of sensitive cultivar. Neither parameter changed by NaCl in resistant cultivar. NaCl treatment decreased water permeability and osmotic potential in both cultivars; sensitive cultivar was more affected. The results extends our previous data that cell membrane properties are different in salt sensitive and resistant genotypes and so cell permeability could be a potential trait indicating salt tolerance.