Growth Medium Conditions Research Articles

English

  A sequential optimization approach based on statistical experimental designs was employed to optimize growth medium and fermentation conditions, in order to improve the antibiotic activity of Xenorhabdus nematophila TB. Tryptone soyptone broth (TSB) was chosen as the original medium for optimization. Glucose and peptone were identified as the optimum carbon and nitrogen sources using single factor method. Peptone, fermentation time, initial pH and inoculum volume were identified as the critical factors which highly influenced the antibiotic activity of X. nematophila TB using Plackett–Burman (PB) design. The critical factors were subsequently optimized to locate their optimum domain using steepest ascent method and were further optimized using central composite design (CCD), involving response surface methodology (RSM). The optimum growth medium and fermentation conditions for antibiotic production by X. nematophila TB consisted of glucose 5.00 g/l, peptone 25.6 g/l, NaCl 5.00 g/l, K2HPO4 2.50 g/l, initial pH 7.59, medium volume (100/250 ml), inoculation age (OD600 nm: 2.00), inoculum volume 9.95%, rotary speed 150 rpm, temperature 25°C and fermentation time 54.1 h. An overall of 73.52% increase in antibiotic activity (418.7 U/ml) was obtained as compared with the unoptimized conditions (241.3 U/ml). This study provides a powerful tool to optimize the bioprocess of the antibiotic production by X. nematophila TB.   Key words: Xenorhabdus nematophila, antibiotic activity, growth medium, fermentation conditions, optimization, response surface methodology.

Polyhydroxybutyrate in Rhizobium and Bradyrhizobium: quantification and phbC gene expression

Polyhydroxyalkanoates (PHAs) are hydroxyalkanoate polymers that are produced and accumulate by many kinds of bacteria. These polymers act as an energy store for bacteria. Polyhydroxybutyrate (PHB) is the most studied polymer in the PHA family. These polymers have awakened interest in the environmental and industrial research areas because they are biodegradable and have thermoplastic qualities, like polypropylene. In this work, we analyzed the PHB production in Bradyrhizobium sp., Rhizobium leguminosarum bv. phaseoli, and Rhizobium huautlense cultured with two different carbon sources. We did biochemical quantification of PHB production during the three phases of growth. Moreover, these samples were used for RNA extraction and phbC gene expression analysis via real-time PCR. The bacteria showed different manner of growth, PHB accumulation and phbC gene expression when different quantity and quality of carbon sources were used. These results showed that under different growth media conditions, the growth and metabolism of different species of bacteria were influenced. These differences reflect the increase or decrease in PHB accumulation.

Effect of Varied Ionic Calcium on Human Adipose-Derived Stem Cell Mineralization

Human adipose-derived stem cells (hASCs) are a relatively abundant and accessible stem cell source with multilineage differentiation capability and have great potential for bone tissue engineering applications. The success of bone tissue engineering is intimately linked with the production of a mineralized matrix that mimics the natural mineral present within native bone. In this study, we examined the effects of ionic calcium levels of 1.8 (normal concentration in cell culture medium), 8, and 16 mM on hASCs seeded in both two-dimensional monolayer and three-dimensional electrospun scaffolds and cultured in either complete growth medium (CGM) or osteogenic differentiation medium (ODM). The impact of calcium supplementation on hASC viability, proliferation, and mineral deposition was determined. hASCs remained viable for all experimental treatments. hASC proliferation increased with the addition of 8 mM Ca(2+) CGM, but decreased for the 16 mM Ca(2+) CGM treatment. Materials deposited by hASCs were analyzed using four techniques: (1) histological staining with Alizarin Red S, (2) calcium quantification, (3) Fourier transform infrared spectroscopy, and (4) wide-angle X-ray diffraction. Mineral deposition was significantly enhanced under both growth and osteogenic medium conditions by increasing extracellular Ca(2+). The greatest mineral deposition occurred in the ODM 8 mM Ca(2+) treatment group. Fourier transform infrared spectroscopy analysis indicated that elevated calcium concentrations of 8 mM Ca(2+) significantly increased both PO(4) amount and PO(4) to protein ratio for ODM. X-ray diffraction indicated that mineral produced with elevated Ca(2+) in both CGM and ODM had a crystalline structure characteristic of hydroxyapatite. Ionic calcium should be considered a potent regulator in hASC mineralization and could serve as a potential treatment for inducing prompt ossification of hASC-seeded scaffolds for bone tissue engineering prior to implantation.

Medium-mediated DNA repair response after ionizing radiation is correlated with the increase of specific cytokines in human fibroblasts

Radiation induced bystander effects, either protective or adverse, have been identified in a variety of cells and for different endpoints. They are thought to arise from communication between cells through direct cell–cell contacts and via transmissible molecules secreted into the medium by targeted cells.We have investigated medium-mediated damage response in human dermal fibroblasts (HDF) after exposure to ionizing irradiation. We show that HDF experience an elevated level of double stranded DNA damage repair response when incubated with conditioned growth medium of irradiated cells. The magnitude of this response is much lower than observed for directly irradiated cells and is proportional to the radiation dose, as is its persistence across time. Since secretion of cytokines is one of the possible pathways linking targeted and non-targeted cells a multiplex analysis was performed. Four cytokines – IL6, IL8, MCP-1 and RANTES – were identified in the growth medium of irradiated cells after exposure to X-rays (2Gy). These cytokines were significantly upregulated and each cytokine showed differential upregulation kinetics. Finally we performed a functional analysis to see if IL6 and MCP-1 could induce γH2AX foci formation. IL6 caused a significant increase in spot occupancy compared to controls. Although only indicative MCP-1 appears to have the opposite effect as it caused a drop in spot occupancy. The combined addition of these two cytokines produced no significant response was observed. Both IL6 and MCP-1 have an effect on the γH2AX spot occupancy possibly linking these cytokines to the bystander response.

Galactoxylomannans from Cryptococcus neoformans Varieties neoformans and grubii Are Structurally and Antigenically Variable

Prior studies have established that the Cryptococcus neoformans capsular polysaccharide component galactoxylomannan (GalXM) manifests serotype-related structural differences that translate into antigenic differences. We analyzed GalXM from acapsular serotype A and D strains by carbohydrate analysis and static and dynamic light scattering to determine mass, effective diameter, polydispersity, and diffusion coefficients. Multiangle laser light scattering showed that GalXM from C. neoformans var. grubii strain cap59 (serotype A) had larger molecular mass (4.21 x 10(6) +/- 0.95 x 10(6) g/mol) and radius of gyration (207 +/- 27 nm) than GalXM from C. neoformans var. neoformans cap67 (serotype D). cap67 GalXM had corresponding values of 0.70 x 10(6) +/- 0.05 x 10(6) g/mol and 120 +/- 22 nm, respectively. The effective diameter for GalXM and polydispersity from the two strains varied depending on temperature and medium growth conditions, indicating that GalXM structure can vary within a strain, depending on its environment. Zeta potential determinations were negative for GalXM from both strains under all conditions, consistent with the recently reported presence of glucuronic acid. These results imply that C. neoformans GalXM, like glucuronoxylomannan, can manifest variety- and growth condition-related variations. Analysis of 16 C. neoformans and 7 Cryptococcus gattii strains with polyclonal antibody to a GalXM strain revealed antigenic similarities among the C. neoformans variety neoformans and grubii strains and no reactivity with C. gattii. As a result of the deleterious effects of GalXM on immune function, structural and antigenic variability between serotypes may translate into differences in immunomodulatory effects.

Optimization of Growth Medium and Enzyme Assay Conditions for Crude Cellulases Produced by a Novel Thermophilic and Cellulolytic Bacterium, Anoxybacillus sp. 527

A newly isolated Anoxybacillus sp. 527 was found to grow on crystalline cellulose as sole carbon and energy sources. Cellulases secreted by strain 527 were better induced by cellobiose, followed by glucose, lactose, sucrose, and cellulose. Cellulase secretion was enhanced by an optimized medium. Cellulase activity was increased by the addition of Ca(2+) and NH (4) (+) and achieved maximum as 7.0 FPU ml(-1) at 70 degrees C and pH 6.0. Even at 100 degrees C, the enzymes were still active, which implies their potential application in large-scale cellulose conversion process.

PICK1-mediated Glutamate Receptor Subunit 2 (GluR2) Trafficking Contributes to Cell Death in Oxygen/Glucose-deprived Hippocampal Neurons

Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca2+/Zn2+-permeable, GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents in hippocampal neurons show marked inward rectification and increased sensitivity to channel blockers selective for GluR2-lacking AMPARs. This occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression and a rapid internalization of GluR2-containing AMPARs during the OGD insult, which are replaced by GluR2-lacking receptors. The mechanisms that underlie this rapid change in subunit composition are unknown. Here, we demonstrate that this trafficking event shares features in common with events that mediate long term depression and long term potentiation and is initiated by the activation of N-methyl-d-aspartic acid receptors. Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death. This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.

Membrane Type-1 Matrix Metalloproteinase Potentiates Basic Fibroblast Growth Factor-Induced Corneal Neovascularization

Corneal neovascularization is one of the leading causes of blindness. The aim of this study was to evaluate the pro-angiogenic role of corneal fibroblast-derived membrane type-1 matrix metalloproteinase (MT1-MMP) on basic fibroblast growth factor (bFGF)-induced corneal neovascularization in vivo and in vitro. Immunohistochemical studies demonstrated that MT1-MMP was expressed in keratocytes and immortalized corneal fibroblast cell lines. Vascular endothelial growth factor protein levels were increased after bFGF-stimulation of wild-type fibroblast cells compared with MT1-MMP knockout fibroblast cells. Corneal vascularization was significantly increased after a combination of bFGF pellet implantation and naked MT1-MMP DNA injection in wild-type mouse corneas compared with either bFGF pellet implantation or naked MT1-MMP DNA-injected corneas. Western blotting analysis of the phosphorylation levels of the key signaling molecules (p38, JNK, and ERK) demonstrated that phosphorylation levels of both p38 and JNK were diminished after bFGF stimulation of MT1-MMP knockout cells compared with wild-type and MT1-MMP knockin cells. These results suggest that MT1-MMP potentiates bFGF-induced corneal neovascularization, likely by modulating the bFGF signal transduction pathway.

Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation.

BackgroundDictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA.ResultsWe find that the extracellular accumulation of AprA increases with cell density and reaches a concentration of 0.3 μg/ml near a stationary cell density. When added to wild-type or aprA- cells, recombinant AprA (rAprA) significantly slows proliferation at 0.1 μg/ml and higher concentrations. From 4 to 64 μg/ml, the effect of rAprA is at a plateau, slowing but not stopping proliferation. The proliferation-inhibiting activity of rAprA is roughly the same as that of native AprA in conditioned growth medium. Proliferating aprA- cells show saturable binding of rAprA to 92,000 ± 11,000 cell-surface receptors with a KD of 0.03 ± 0.02 μg/ml. There appears to be one class of binding site, and no apparent cooperativity. Native AprA inhibits the binding of rAprA to aprA- cells with a Ki of 0.03 μg/ml, suggesting that the binding kinetics of rAprA are similar to those of native AprA. The proliferation of cells lacking CrlA, a cAMP receptor-like protein, or cells lacking CfaD are not affected by rAprA. Surprisingly, both cell types still bind rAprA.ConclusionTogether, the data suggest that AprA functions as an autocrine proliferation-inhibiting factor by binding to cell surface receptors. Although AprA requires CfaD for activity, it does not require CfaD to bind to cells, suggesting the possibility that cells have an AprA receptor and a CfaD receptor, and activation of both receptors is required to slow proliferation. We previously found that crlA- cells are sensitive to CfaD. Combined with the results presented here, this suggests that CrlA is not the AprA or CfaD receptor, and may be the receptor for an unknown third factor that is required for AprA and CfaD activity.

Quantitative Analysis of Weissella cibaria against Periodontopathic Bacteria by Real-time PCR

The objective of this study was to analyze quantitatively whether Weissella cibaria could affect the proliferation of five periodontopathic bacteria, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum, after incubation for 8~48 h. In addition, by using real-time PCR with a dual-labeled probe, each growth of bacteria was examined under different growth media conditions. The proliferation of periodontopathic bacteria was significantly inhibited by W. cibaria after incubation for 24~48 h (p ＜ 0.05), whereas the growth of W. cibaria was not affected by these pathogenic bacteria. The growth of P. gingivalis, T. forsythia and T. denticola significantly increased in each growth media after incubation for 24 h (p ＜ 0.05), as compared to the culture in mixed growth media. However, no differences in the growth of five periodontopathic bacteria were observed between each growth media and mixed media after incubation for 48 h. The growth and pH of W. cibaria culture significantly were changed in MRS after incubation for 24~48 h (p ＜ 0.05), as compared to the bacterial culture in mixed growth media. The pH of P. gingivalis and F. nucleatum culture significantly was changed in both growth media and mixed media after incubation for 24~48 h (p ＜ 0.05). Our data indicate that W. cibaria significantly inhibits the proliferation of five periodontopathic bacteria and each growth of bacteria is quantitatively analyzed under various media conditions by real-time PCR.

Expression profiling of Streptomyces peucetius metabolic genes using DNA microarray analysis

Doxorubicin (DXR) and daunorubicin (DNR) are anthracycline antibiotics produced by Streptomyces peucetius and widely used as cancer chemotherapeutic agents. To improve their productivity, regulation of DXR/DNR synthesis genes as well as central metabolic pathway genes must be understood more clearly. So far, studies have focused on DXR/DNR gene regulation. To investigate the correlation between the central metabolic pathway genes and DXR/DNR productivity, we selected 265 genes involved in glycolysis, fermentation, the citric acid cycle, butanoate metabolism, etc., and searched for their sequences in the S. peucetius genome by comparing gene sequences to those of Streptomyces coelicolor. The homologous genes were amplified by PCR and arrayed on glass microarray slides. Gene expression was monitored under two different growth media conditions, R2YE and NDYE. Genes involved in the production of malonyl-CoA and propionyl-CoA, the main precursors for doxorubicin synthesis, were mainly upregulated in NDYE media. Genes related to acetyl-CoA and the urea cycle were also upregulated. These changes in gene expression were confirmed by real-time RT-PCR.

Increased Secretion and Expression of Myostatin in Skeletal Muscle From Extremely Obese Women

OBJECTIVE—Obesity is associated with endocrine abnormalities that predict the progression of insulin resistance to type 2 diabetes. Because skeletal muscle has been shown to secrete proteins that could be used as biomarkers, we characterized the secreted protein profile of muscle cells derived from extremely obese (BMI 48.8 ± 14.8 kg/m2; homeostasis model assessment [HOMA] 3.6 ± 1.0) relative to lean healthy subjects (BMI 25.7 ± 3.2 kg/m2; HOMA 0.8 ± 0.2).RESEARCH DESIGN AND METHODS—We hypothesized that skeletal muscle would secrete proteins that predict the severity of obesity. To test this hypothesis, we used a “bottom-up” experimental design using stable isotope labeling by amino acids in culture (SILAC) and liquid chromatography/mass spectometry/mass spectometry (LC-MS/MS) to both identify and quantify proteins secreted from cultured myotubes derived from extremely obese compared with healthy nonobese women.RESULTS—Using SILAC, we discovered a 2.9-fold increase in the secretion of myostatin from extremely obese human myotubes. The increased secretion and biological activity of myostatin were validated by immunoblot (3.16 ± 0.18, P < 0.01) and a myoblast proliferation assay using conditioned growth medium. Myostatin was subsequently shown to increase in skeletal muscle (23%, P < 0.05) and plasma (35%, P < 0.05) and to correlate (r2 = 0.6, P < 0.05) with the severity of insulin resistance.CONCLUSIONS—Myostatin is a potent antianabolic regulator of muscle mass that may also play a role in energy metabolism. These findings show that increased expression of myostatin in skeletal muscle with obesity and insulin resistance results in elevated circulating myostatin. This may contribute to systemic metabolic deterioration of skeletal muscle with the progression of insulin resistance to type 2 diabetes.

The secretedDictyosteliumprotein CfaD is a chalone

Dictyostelium discoideum cells secrete CfaD, a protein that is similar to cathepsin proteases. Cells that lack cfaD proliferate faster and reach a higher stationary-phase density than wild-type cells, whereas cells that overexpress CfaD proliferate slowly and reach the stationary phase when at a low density. On a per-nucleus basis, CfaD affects proliferation but not growth. The drawback of not having CfaD is a reduced spore viability. Recombinant CfaD has no detectable protease activity but, when added to cells, inhibits the proliferation of wild-type and cfaD(-) cells. The secreted protein AprA also inhibits proliferation. AprA is necessary for the effect of CfaD on proliferation. Molecular-sieve chromatography indicates that in conditioned growth medium, the 60 kDa CfaD is part of a approximately 150 kDa complex, and both chromatography and pull-down assays suggest that CfaD interacts with AprA. These results suggest that two interacting proteins may function together as a chalone signal in a negative feedback loop that slows Dictyostelium cell proliferation.

LIM Kinase and Slingshot Are Critical for Neurite Extension

Cofilin and its closely related protein, actin-depolymerizing factor (ADF), are key regulators of actin cytoskeleton dynamics that have been implicated in growth cone motility and neurite extension. Cofilin/ADF are inactivated by LIM kinase (LIMK)-catalyzed phosphorylation and reactivated by Slingshot (SSH)-catalyzed dephosphorylation. Here we examined the roles of cofilin/ADF, LIMKs (LIMK1 and LIMK2), and SSHs (SSH1 and SSH2) in nerve growth factor (NGF)-induced neurite extension. Knockdown of cofilin/ADF by RNA interference almost completely inhibited NGF-induced neurite extension from PC12 cells, and double knockdown of SSH1/SSH2 significantly suppressed both NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, thus indicating that cofilin/ADF and their activating phosphatases SSH1/SSH2 are critical for neurite extension. Interestingly, NGF stimulated the activities of both LIMK1 and LIMK2 in PC12 cells, and suppression of LIMK1/LIMK2 expression or activity significantly reduced NGF-induced neurite extension from PC12 cells or chick dorsal root ganglion (DRG) neurons. Inhibition of LIMK1/LIMK2 activity reduced actin filament assembly in the peripheral region of the growth cone of chick DRG neurons. These results suggest that proper regulation of cofilin/ADF activities through control of phosphorylation by LIMKs and SSHs is critical for neurite extension and that LIMKs regulate actin filament assembly at the tip of the growth cone.

Mechanisms of Transcription Activation Exerted by GadX and GadW at the gadA and gadBC Gene Promoters of the Glutamate-Based Acid Resistance System in Escherichia coli

In Escherichia coli the gad system protects the cell from the extreme acid stress encountered during transit through the host stomach. The structural genes gadA, gadB, and gadC encode two glutamate decarboxylase isoforms and a glutamate/gamma-aminobutyrate (GABA) antiporter, respectively. Glutamate decarboxylation involves both proton consumption and production of GABA, a neutral compound which is finally exported via the GadC antiporter. Regulation of gadA and gadBC transcription is very complex, involving several circuits controlling expression under different growth phase, medium, and pH conditions. In this study we found that the AraC-like activators GadX and GadW share the same 44-bp binding sites in the gadA and gadBC regulatory regions. The common binding sites are centered at 110.5 bp and 220.5 bp upstream of the transcriptional start points of the gadA and gadBC genes, respectively. At the gadA promoter this regulatory element overlaps one of the binding sites of the repressor H-NS. The DNA of the gadBC promoter has an intrinsic bend which is centered at position -121. These findings, combined with transcriptional regulation studies, may account for the two different mechanisms of transcriptional activation by GadX and GadW at the two promoters studied. We speculate that while at the gadA promoter GadX and GadW activate transcription by displacing H-NS via an antirepressor mechanism, at the gadBC promoter the mechanism of activation involves looping of the DNA sequence between the promoter and the activator binding site.

Bioluminescence of Vibrio fischeri in continuous culture: Optimal conditions for stability and intensity of photoemission

Continuous cultivation of the bioluminescent bacterium, Vibrio fischeri NRRL-B-11177, in a fermenter provided a constant supply of bacteria in exponential growth phase. These bacteria may be used as stable bioindicators to measure perturbation of the metabolic state by physical and chemical agents. However, optimization of light emission necessitates careful choice of growth medium and culture operating conditions. Optimized conditions that supported long-term cultivation of V. fischeri NRRL-B-11177 with stable intense bioluminescence were: a dilution rate of 0.08–0.09 h − 1 , air supply of 600 ml min − 1 , stirring of 300–350 rpm at a constant incubation temperature within the range of 20 to 26 °C and pH 7.8. These conditions were as successful in a 500-ml as in a 10-ml fermenter. This system provided a reliable long-term (more than 1 month) continuous culture facility for the reproducible measurement of perturbation of V. fischeri by monitoring changes in luminescence.

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 microL and photomicrographed. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10(2) promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

Preliminary results on nematicidal activity from culture filtrates of Basidiomycetes against the pine wood nematode,Bursaphelenchus xylophilus (Aphelenchoididae)

One hundred and eighty one fungal species that were isolated from the fresh fruiting bodies collected in the Mountains of Pu Er County of Yunnan Province, China were tested on the pine wood nematode,Bursaphelenchus xylophilus in vitro. Each fungal species was grown in Czapek broth and potato dextrose broth (PDB). Fifteen filtrates fromAmauroderma austrosinense, Amauroderma macer, Filoboletus sp.,Laccaria tortilis, Lactarius gerardii, Lentinula edodes, Oudemansiella longipes, Oudemansiella mucida, Peziza sp.,Pleurotus sp.,Sinotermitomyces carnosus (two strains),Strobilomyces floccopus, Termitomyces albuminosus, Tylopilus striatulus grown on PDB were found to be pathogenic to the tested nematodes. Eleven filtrates fromAmanita junguillea, Amanita sp.,Daedalea sepiaria, Fistulina hepatica, Omphalotus olearius, Oudemansiella mucida, Peziza sp.,Pleurotus pulmatus, Ramaria sp.,Tricholoma conglobatum, Tylopilus striatulus grown on Czapek broth were also pathogenic to the nematodes. When screening for nematicidal potential of fungi, it is important to study the growth medium conditions necessary to obtain the optimal nematicidal effect as fungal filtrates growing on different liquid media showed a very inconsistent toxicity towards nematodes.

The structural bases of long-term anabiosis in non-spore-forming bacteria

Peculiarities of the structural organization in non-spore-forming bacteria associated with long-term anabiosis were revealed both in laboratory cultures and in natural populations isolated from 1–3-Myr-old Eastern Siberian permafrost and tundra soil. Different advanced methods were used, including (a) high-resolution electron microscopy; (b) simulation of in situ conditions in the laboratory by varying the composition of growth medium and cultivation conditions; (c) low-temperature fractionation to isolate and concentrate microbial cells from natural soils; (d) comparative morphological analysis of microbial cells in model cultures and natural soils (in situ). Under laboratory conditions, the intense formation of resting cells by representatives of various taxa of eubacteria and halophilic archaea occurred in 2–9-month-old cultures grown in carbon-, nitrogen-, or phosphorus-limited media, in starved cell suspensions in the presence of sodium silicate, or on soil agar. Among resting cells, we revealed cystlike forms having a complicated structure and common features. These included a thick capsule; a thickened and multiprofile cell wall; the presence of large intramembrane particles on PF- and EF-fracture surfaces; fine-grained or lumpy cytoplasm; and a condensed nucleoid. The general morphological properties, ultrastructural organization, physiological features of cystlike cells, and their ability to germinate under the appropriate conditions suggest the existence of constitutive dormancy in non-spore-forming bacteria. It was found that the majority of microorganisms in permafrost and tundra soil are cystlike cells, very similar to those in laboratory cultures. Anabiotic (resting) cystlike cells are responsible for the survival of non-spore-formers in extreme Earth habitats and may be regarded as possible analogs of extraterrestrial forms of microbial life.

Candida albicans -Conditioned Medium Protects Yeast Cells from Oxidative Stress: a Possible Link between Quorum Sensing and Oxidative Stress Resistance

Candida albicans, the most frequent fungal pathogen of humans, encounters high levels of oxidants following ingestion by professional phagocytes and through contact with hydrogen peroxide-producing bacteria. In this study, we provide evidence that C. albicans is able to coordinately regulate the oxidative stress response at the global cell population level by releasing protective molecules into the surrounding medium. We demonstrate that conditioned medium, which is defined as a filter-sterilized supernatant from a C. albicans stationary-phase culture, is able to protect yeast cells from both hydrogen peroxide and superoxide anion-generating agents. Exponential-phase yeast cells preexposed to conditioned medium were able to survive levels of oxidative stress that would normally kill actively growing yeast cells. Heat treatment, digestion with proteinase K, pH adjustment, or the addition of the oxidant scavenger alpha-tocopherol did not alter the ability of conditioned medium to induce a protective response. Farnesol, a heat-stable quorum-sensing molecule (QSM) that is insensitive to proteolytic enzymes and is unaffected by pH extremes, is partly responsible for this protective response. In contrast, the QSM tyrosol did not alter the sensitivity of C. albicans cells to oxidants. Relative reverse transcription-PCR analysis indicates that Candida-conditioned growth medium induces the expression of CAT1, SOD1, SOD2, and SOD4, suggesting that protection may be mediated through the transcriptional regulation of antioxidant-encoding genes. Together, these data suggest a link between the quorum-sensing molecule farnesol and the oxidative stress response in C. albicans.