Stationary Phase Of Cell Growth Research Articles

Key interactions of RNA polymerase with 6S RNA and secondary channel factors during pRNA synthesis

Small non-coding 6S RNA mimics DNA promoters and binds to the σ70 holoenzyme of bacterial RNA polymerase (RNAP) to suppress transcription of various genes mainly during the stationary phase of cell growth or starvation. This inhibition can be relieved upon synthesis of short product RNA (pRNA) performed by RNAP from the 6S RNA template. Here, we have shown that pRNA synthesis depends on specific contacts of 6S RNA with RNAP and interactions of the σ finger with the RNA template in the active site of RNAP, and is also modulated by the secondary channel factors. We have adapted a molecular beacon assay with fluorescently labeled σ70 to analyze 6S RNA release during pRNA synthesis. We found the kinetics of 6S RNA release to be oppositely affected by mutations in the σ finger and in the CRE pocket of core RNAP, similarly to the reported role of these regions in promoter-dependent transcription. Secondary channel factors, DksA and GreB, inhibit pRNA synthesis and 6S RNA release from RNAP, suggesting that they may contribute to the 6S RNA-mediated switch in transcription during stringent response. Our results demonstrate that pRNA synthesis depends on a similar set of contacts between RNAP and 6S RNA as in the case of promoter-dependent transcription initiation and reveal that both processes can be regulated by universal transcription factors acting on RNAP.

Nutrient limitation and oxidative stress induce the promoter of acetate operon in Salmonella Typhimurium.

Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the β-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in β galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments.

New insights into the influence of pre-culture on robust solvent production of C. acetobutylicum

Clostridia are known for their solvent production, especially the production of butanol. Concerning the projected depletion of fossil fuels, this is of great interest. The cultivation of clostridia is known to be challenging, and it is difficult to achieve reproducible results and robust processes. However, existing publications usually concentrate on the cultivation conditions of the main culture. In this paper, the influence of cryo-conservation and pre-culture on growth and solvent production in the resulting main cultivation are examined. A protocol was developed that leads to reproducible cultivations of Clostridium acetobutylicum. Detailed investigation of the cell conservation in cryo-cultures ensured reliable cell growth in the pre-culture. Moreover, a reason for the acid crash in the main culture was found, based on the cultivation conditions of the pre-culture. The critical parameter to avoid the acid crash and accomplish the shift to the solventogenesis of clostridia is the metabolic phase in which the cells of the pre-culture were at the time of inoculation of the main culture; this depends on the cultivation time of the pre-culture. Using cells from the exponential growth phase to inoculate the main culture leads to an acid crash. To achieve the solventogenic phase with butanol production, the inoculum should consist of older cells which are in the stationary growth phase. Considering these parameters, which affect the entire cultivation process, reproducible results and reliable solvent production are ensured.Key points• Both cryo- and pre-culture strongly impact the cultivation of C. acetobutylicum• Cultivation conditions of the pre-culture are a reason for the acid crash• Inoculum from cells in stationary growth phase ensures shift to solventogenesisGraphical

Gene rppA co-regulated by LRR, SigA, and CcpA mediates antibiotic resistance in Bacillus thuringiensis.

Antibiotic resistance genes are usually tightly controlled by transcription factors and RNA regulatory elements including sRNAs, riboswitches, and attenuators, and their expression is activated to respond to antibiotic exposure. In previous work, we revealed that the rppA gene is regulated by attenuator LRR and two mistranslation products in Bacillus thuringiensis BMB171. However, its function and promoter regulation is still not precise. In this study, we demonstrated that the encoding product of the rppA gene acts as an ARE1 ABC-F protein and confers resistance to antibiotics virginiamycin M1 and lincomycin when overexpressed. Besides the reported attenuator LRR, the expression of the rppA gene is controlled by the sigma factor SigA and a global transcription factor CcpA. Consequently, its promoter activity is mainly maintained at the stationary phase of cell growth and inhibited in the presence of glucose. Our study revealed the function and regulation of the rppA gene in detail. KEY POINTS: • The RppA protein acts as an ARE1 ABC-F protein • The rppA gene confers resistance to antibiotics virginiamycin M1 and lincomycin when overexpressed • The expression of the rppA gene is regulated by the sigma factor SigA and the pleiotropic regulator CcpA.

Two Novel Semisynthetic Lipoglycopeptides Active against Staphylococcus aureus Biofilms and Cells in Late Stationary Growth Phase.

The increase in antibiotic resistance among Gram-positive bacteria underscores the urgent need to develop new antibiotics. New antibiotics should target actively growing susceptible bacteria that are resistant to clinically accepted antibiotics including bacteria that are not growing or are protected in a biofilm environment. In this paper, we compare the in vitro activities of two new semisynthetic glycopeptide antibiotics, MA79 and ERJ390, with two clinically used glycopeptide antibiotics—vancomycin and teicoplanin. The new antibiotics effectively killed not only exponentially growing cells of Staphylococcus aureus, but also cells in the stationary growth phase and biofilm.

Heat resistance, membrane fluidity and sublethal damage in Staphylococcus aureus cells grown at different temperatures

In this work the influence of growth temperature (10–42 °C) on Staphylococcus aureus heat resistance was studied, and its relationship with the ability of cells to repair sublethal damages and with membrane fluidity was evaluated. Non-linear, convex from above survival curves were obtained, and therefore a special case of the Baranyi model was used to fit them. For exponential phase cells, heat resistance did not change with growth temperature in the range between 10 and 37 °C, but cells grown at 42 °C were significantly more resistant, showing D58 and shoulder length (sl58) values 2.5 and 4 times greater than the others, respectively. For stationary growth phase cells, an increase in growth temperature above 20 °C resulted in an increase in D58 values, and cells grown at 42 °C also displayed the highest D58 and sl58 values. The increased heat resistance at 58 °C of stationary growth phase cells grown at higher temperatures was coincident with the appearance of a higher proportion of sublethally damaged cells capable of recovery and outgrowth in non-selective medium. Membrane fluidity was measured at treatment temperatures, and it was observed that those cells with more rigid membranes displayed greater heat resistance (Pearson coefficient = 0.969***). Additionally, S. aureus cells whose membrane was fluidized through exposure to benzyl alcohol were notably sensitized against the action of heat, in a concentration-dependent manner. Results obtained in this research indicate that membrane physical state could be an important factor determining the survival capacity of bacterial cells to a heat treatment.

Impairment of the Viability of Transformed Chinese Hamster Cells in a Nonsubcultured Culture under the Influence of Exogenous Oxidized Guanoside is Manifested Only in the Stationary Phase of Growth

Despite the fact that oxidation products of nucleotides and nucleosides are markers of oxidative stress, reports of the paradoxical ability of these compounds to protect cells from the harmful effects of reactive oxygen species began to appear more often. Among all nitrogenous bases, guanine is most susceptible to the influence of oxidative stress; therefore, guanosine is oxidized more often than other bases. In the present work, the effect of exogenous 8-oxo-2′-deoxyguanosine on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during cell proliferation slowing down within a single passage and subsequent “aging” in the stationary growth phase) of nonsubcultured transformed Chinese hamster cells was studied. We showed that the nucleoside is rapidly absorbed by the cells from the medium, but it does not affect the growth of the culture, and impairs the viability of the cells in the late stationary growth phase. Thus, no mitogenic or geroprotective effect of 8-oxo-2′-deoxyguanosine was found.

Enhancing cadmium bioremediation by a complex of water-hyacinth derived pellets immobilized with Chlorella sp.

A complex of water-hyacinth derived pellets immobilized with Chlorella sp. was applied, for the first time, in the bioremediation of Cadmium (Cd). The Cd(II) removal efficiency of the complex was optimized by investigating several parameters, including the pellet materials, algal culture age, and light intensity. Results showed that the Cd(II) removal efficiency was positively related to the algal immobilization efficiency and the algal bioaccumulation capacity. Since higher surface hydrophilicity leads to higher immobilization efficiency, the water-hyacinth leaf biochar pellet (WLBp) was selected as the optimal carrier. A maximum Cd(II) removal efficiency of 92.45% was obtained by the complex of WLBp immobilized with algal cells in stationary growth phase and illuminated with a light intensity of 119 μmol m−2 s−1. Recovery tests on both microalgal cells and the WLBp demonstrated that the algal cells and the biochar pellet can be economically recycled and reused.

Photodynamic damage predominates on different targets depending on cell growth phase of Candida albicans

Photodynamic inactivation (PDI) has been reported to be effective to eradicate a wide variety of pathogens, including antimicrobial-resistant microorganisms. The aim of this study was to identify the potential molecular targets of PDI depending on growth phase of Candida albicans. Fungal cells in lag (6h) and stationary (48h) phases were submitted to PDI mediated by methylene blue (MB) combined with a (662±21) nm-LED, at 360mW of optical power. Pre-irradiation time was 10min and exposure times were 12min, 15min and 18min delivering radiant exposures of 129.6J/cm2, 162J/cm2 and 194.4J/cm2, respectively, on a 24-well plate of about 2cm2 at an irradiance of 180mW/cm2. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force spectroscopy (AFS) and Fourier transform infrared spectroscopy (FT-IR) were employed to evaluate the photodynamic effect in young and old fungal cells following 15min of irradiation. Morphological analysis revealed wrinkled and shrunk fungal cell membrane for both growth phases while extracellular polymeric substance (EPS) removal was only observed for old fungal cells. Damaged intracellular structures were more pronounced in young fungal cells. The surface nanostiffness of young fungal cells decreased after PDI but increased for old fungal cells. Cellular adhesion force was reduced for both growth phases. Fungal cells in lag phase predominantly showed degradation of nucleic acids and proteins, while fungal cells in stationary phase showed more pronounced degradation of polysaccharides and lipids. Taken together, our results indicate different molecular targets for fungal cells in lag and stationary growth phase following PDI.

Pseudo-nitzschia simulans sp. nov. (Bacillariophyceae), the first domoic acid producer from Chinese waters

The genus Pseudo-nitzschia has attracted attention because of production of the toxin, domoic acid (DA), causing Amnesic Shellfish Poisoning (ASP). Pseudo-nitzschia blooms occur frequently in Chinese coastal waters, and DA has been detected in several marine organisms, but so far no Pseudo-nitzschia strains from Chinese waters have been shown to produce DA. In this study, monoclonal Pseudo-nitzschia strains were established from Chinese coastal waters and examined using light microscopy, electron microscopy and molecular markers. Five strains, sharing distinct morphological and molecular features differentiating them from other Pseudo-nitzschia species, represent a new species, Pseudo-nitzschia simulans sp. nov. Morphologically, the taxon belongs to the P. pseudodelicatissima group, cells possessing a central nodule and each stria comprising one row of poroids. The new species is characterized by the poroid structure, which typically comprises two sectors, each sector located near opposite margins of the poroid. The production of DA was examined by liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses of cells in stationary growth phase. Domoic acid was detected in one of the five strains, with concentrations around 1.05–1.54 fg cell−1. This is the first toxigenic diatom species reported from Chinese waters.

Cysteine Addition Promotes Sulfide Production and 4-Fold Hg(II)-S Coordination in Actively Metabolizing Escherichia coli.

The bacterial uptake of mercury(II), Hg(II), is believed to be energy-dependent and is enhanced by cysteine in diverse species of bacteria under aerobic and anaerobic conditions. To gain insight into this Hg(II) biouptake pathway, we have employed X-ray absorption spectroscopy (XAS) to investigate the relationship between exogenous cysteine, cellular metabolism, cellular localization, and Hg(II) coordination in aerobically respiring Escherichia coli (E. coli). We show that cells harvested in exponential growth phase consistently display mixtures of 2-fold and 4-fold Hg(II) coordination to sulfur (Hg-S2 and Hg-S4), with added cysteine enhancing Hg-S4 formation. In contrast, cells in stationary growth phase or cells treated with a protonophore causing a decrease in cellular ATP predominantly contain Hg-S2, regardless of cysteine addition. Our XAS results favor metacinnabar (β-HgS) as the Hg-S4 species, which we show is associated with both the cell envelope and cytoplasm. Additionally, we observe that added cysteine abiotically oxidizes to cystine and exponentially growing E. coli degrade high cysteine concentrations (100-1000 μM) into sulfide. Thermodynamic calculations confirm that cysteine-induced sulfide biosynthesis can promote the formation of dissolved and particulate Hg(II)-sulfide species. This report reveals new complexities arising in Hg(II) bioassays with cysteine and emphasizes the need for considering changes in chemical speciation as well as growth stage.

Pressure-induced inactivation of bacteria through pressure-assisted thawing using a thermal buffer zone.

Abstract Effect of thermal buffer zone was examined on the microbial inactivation through a pressure-assisted thawing. A plastic bag of bacterial suspension enclosed with a thermal buffer zone was frozen at − 50 °C, and treated for 20 min with a pressure-assisted thawing in water of 4 °C. A reduction of 8-log cycle was obtained at 200 MPa for the stationary growth phase cells of Escherichia coli that was suspended in 1% skim milk and enclosed with wheat flour/water paste and two polytetrafluoroethylene plates. When 100% ethanol was used as a thermal buffer and the samples were pressured at 194 MPa in 1% skim milk, levels of E. coli and Listeria monocytogenes were reduced by 6-log cycle and 7-log cycle, respectively. Staphylococcus aureus decreased by 4-log cycle. Industrial relevance This work will contribute to new developments in the pressure processing of foods, since the use of a thermal buffer zone in pressure-assisted thawing was very effective in enhancing the level of pressure-induced microbial inactivation.

LapF and Its Regulation by Fis Affect the Cell Surface Hydrophobicity of Pseudomonas putida.

The ability of bacteria to regulate cell surface hydrophobicity is important for the adaptation to different environmental conditions. The hydrophobicity of cell surface can be determined by several factors, including outer membrane and surface proteins. In this study, we report that an adhesin LapF influences cell surface hydrophobicity of Pseudomonas putida. Cells lacking LapF are less hydrophobic than wild-type cells in stationary growth phase. Moreover, the overexpression of the global regulator Fis decreases surface hydrophobicity by repressing the expression of lapF. Flow cytometry analysis revealed that bacteria producing LapF are more viable when confronted with methanol (a hydrophilic compound) but are more susceptible to 1-octanol (a hydrophobic compound). Thus, these results revealed that LapF is the hydrophobicity factor for the cell surface of P. putida.

Investigation of Growth Phase-Dependent Acid Tolerance in Bifidobacteria longum BBMN68.

The underlying mechanisms imparting the growth phase-dependent acid tolerance have not been extensively investigated. In this study, we compared the acid resistance of the Bifidobacterium longum strain BBMN68 from different growth phases at lethal pH values (pH 2.5, 3.0, and 3.5), and analyzed the activity of H(+)-ATPase, the composition of fatty acids, and the mRNA abundance of ffh, uvrA, recA, lexA, groES, and dnaK in cells from different growth phases. The results indicated that the survival rates of cells from early stationary (ES) and late stationary (LS) growth phases at lethal pH values were significantly higher than those of exponential growth phase cells. Our findings indicated that by inducing a continuously auto-acidizing environment during cell growth, the acid resistance of ES and LS cells was strengthened. The higher activity of H(+)-ATPase, the decrease in unsaturated fatty acids, and the increased expression of genes involved in DNA repair and protein protection in the cells in stationary growth phase were all implicated in the significantly increased acid resistance of ES and LS cells compared with exponential growth phase cells of the B. longum strain BBMN68.

Optimization of submerged Aspergillus oryzae S2 α-amylase production.

Use of 4 agro-industrial by products and organic materials as nitrogen sources for production of Aspergillus oryzae S2 α-amylase in liquid culture was investigated. The 2 agro-industrial byproducts maltose and saccharose, and also lactose and starch were individually evaluated for use as carbon sources. A Box-Behnken experimental design was used to determine optimal conditions for production of α-amylase. A maximum amylase activity of 750 U/mL was obtained at a temperature of 24°C, a urea concentration of 1 g/L, and a C/N ratio of 2. Laboratory scale application of optimal conditions in a 7 L fermentor produced a final α-amylase activity of 770 U/mL after 3 days of batch cultivation. Addition of 10% starch to the culture medium each 12 h immediately after the stationary phase of cell growth led to a production yield of 1,220 U/mL at the end of fed-batch cultivation.

Low-level lasers affect Escherichia coli cultures in hyperosmotic stress

Physical characteristics and practical properties have made lasers of interest for biomedical applications. Effects of low-level lasers on biological tissues could occur or be measurable depending on cell type, presence of a pathologic process or whether the cells are in an adverse environment. The objective of this work was to evaluate the survival, morphology and filamentation of E. coli cells proficient and deficient in the repair of oxidative DNA lesions exposed low-level red and infrared lasers submitted to hyperosmotic stress. Wild type and endonuclease VIII deficient E. coli cells in exponential and stationary growth phase were exposed to red and infrared lasers and submitted to hyperosmotic stress. Cell viability, filamentation phenotype and cell morphology were evaluated. Cell viability was not significantly altered but previous laser exposure induced filamentation and an altered area of stressed cells depending on physiologic condition and presence of the DNA repair. Results suggest that previous exposure to low-level red and infrared lasers could not affect viability but induced morphologic changes in cells submitted to hyperosmotic stress depending on physiologic conditions and repair of oxidative DNA lesions.

Quantitative Analysis of Carbon Flow into Photosynthetic Products Functioning as Carbon Storage in the Marine Coccolithophore, Emiliania huxleyi

The bloom-forming coccolithophore Emiliania huxleyi (Haptophyta) is a dominant marine phytoplankton, cells of which are covered with calcareous plates (coccoliths). E. huxleyi produces unique lipids of C37–C40 long-chain ketones (alkenones) with two to four trans-unsaturated bonds, β-glucan (but not α-glucan) and acid polysaccharide (AP) associated with the morphogenesis of CaCO3 crystals in coccoliths. Despite such unique features, there is no detailed information on the patterns of carbon allocation into these compounds. Therefore, we performed quantitative estimation of carbon flow into various macromolecular products by conducting 14C-radiotracer experiments using NaH14CO3 as a substrate. Photosynthetic 14C incorporation into low molecular-mass compounds (LMC), extracellular AP, alkenones, and total lipids except alkenones was estimated to be 35, 13, 17, and 25 % of total 14C fixation in logarithmic growth phase cells and 33, 19, 18, and 18 % in stationary growth phase cells, respectively. However, less than 1 % of 14C was incorporated into β-glucan in both cells. 14C-mannitol occupied ca. 5 % of total fixed 14C as the most dominant LMC product. Levels of all 14C compounds decreased in the dark. Therefore, alkenones and LMC (including mannitol), but not β-glucan, function in carbon/energy storage in E. huxleyi, irrespective of the growth phase. Compared with other algae, the low carbon flux into β-glucan is a unique feature of carbon metabolism in E. huxelyi.Electronic supplementary materialThe online version of this article (doi:10.1007/s10126-015-9632-1) contains supplementary material, which is available to authorized users.

Insights into Vibrio parahaemolyticus CHN25 response to artificial gastric fluid stress by transcriptomic analysis.

Vibrio parahaemolyticus is the causative agent of food-borne gastroenteritis disease. Once consumed, human acid gastric fluid is perhaps one of the most important environmental stresses imposed on the bacterium. Herein, for the first time, we investigated Vibrio parahaemolyticus CHN25 response to artificial gastric fluid (AGF) stress by transcriptomic analysis. The bacterium at logarithmic growth phase (LGP) displayed lower survival rates than that at stationary growth phase (SGP) under a sub-lethal acid condition (pH 4.9). Transcriptome data revealed that 11.6% of the expressed genes in Vibrio parahaemolyticus CHN25 was up-regulated in LGP cells after exposed to AGF (pH 4.9) for 30 min, including those involved in sugar transport, nitrogen metabolism, energy production and protein biosynthesis, whereas 14.0% of the genes was down-regulated, such as ATP-binding cassette (ABC) transporter and flagellar biosynthesis genes. In contrast, the AGF stress only elicited 3.4% of the genes from SGP cells, the majority of which were attenuated in expression. Moreover, the number of expressed regulator genes was also substantially reduced in SGP cells. Comparison of transcriptome profiles further revealed forty-one growth-phase independent genes in the AGF stress, however, half of which displayed distinct expression features between the two growth phases. Vibrio parahaemolyticus seemed to have evolved a number of molecular strategies for coping with the acid stress. The data here will facilitate future studies for environmental stresses and pathogenicity of the leading seafood-borne pathogen worldwide.

Radiosensitivity, liquid-holding recovery and relative biological effectiveness of densely-ionizing radiation after repeated irradiation of yeast cells

Experimental results described earlier showed significantly larger relative biological effectiveness (RBE) values for wild-type diploid cells in comparison with radiosensitive mutants. This aspect was further studied in this paper. Diploid yeast cells were irradiated with gamma rays from 60Co and alpha particles from 239Pu in the stationary phase of cell growth. Survival curves and the kinetics of the liquid-holding recovery were measured. When the irradiated cells had completely recovered from potentially lethal damage, they were again exposed to radiation and allowed post-irradiation recovery. The procedure was repeated three times. By use of a quantitative approach – describing the process of recovery as a decrease in the effective radiation dose –, the probability of recovery per unit time and the proportion of irreversibly damaged cells were quantitatively estimated. It was shown that the irreversible fraction of cell injury was increased after repeated exposures to gamma rays, from 0.4 after the first irradiation to 0.7 after the third exposure. The effect was more clearly expressed after exposure to densely ionizing radiation, the corresponding values being 0.5 and 1.0. In contrast, the recovery constant did not depend on the number of repeated irradiations and only slightly depended on radiation quality. It is suggested that the process of recovery from potentially lethal radiation damage itself is not impaired after repeated exposures to both low- and high-LET radiations, and the decrease in the ability of the cell to recover from radiation damage is mainly explained by the increase in the proportion of irreversibly damaged cells.

Metabolic and transcriptional activities of Staphylococcus aureus challenged with high-doses of daptomycin

Treatment of stationary growth phase Staphylococcus aureus SA113 with 100-fold of the MIC of the lipopeptide antibiotic daptomycin leaves alive a small fraction of drug tolerant albeit genetically susceptible bacteria. This study shows that cells of this subpopulation exhibit active metabolism even hours after the onset of the drug challenge. Isotopologue profiling using fully 13C-labeled glucose revealed de novo biosynthesis of the amino acids Ala, Asp, Glu, Ser, Gly and His. The isotopologue composition in Asp and Glu suggested an increased activity of the TCA cycle under daptomycin treatment compared to unaffected stationary growth phase cells. Microarray analysis showed differential expression of specific genes 10min and 3h after addition of the drug. Besides factors involved in drug response, a number of metabolic genes appear to shape the signature of daptomycin-tolerant S. aureus cells. These observations will be useful toward the development of new strategies against persisters and related forms of bacterial cells with downshifted physiology.