The bacterium Xenorhabdus nematophilus depresses nodulation reactions to infection by inhibiting eicosanoid biosynthesis in tobacco hornworms,Manduca sexta

The bacterium, Xenorhabdus nematophilus, is a virulent insect pathogen. We tested the hypothesis that this bacterium impairs insect cellular immune defense reactions by inhibiting biosynthesis of eicosanoids involved in mediating cellular defense reactions. Fifth instar tobacco hornworms, Manduca sexta, produced melanized nodules in reaction to challenge with living and heat-killed X. nematophilus. However, the nodulation reactions were much attenuated in insects challenged with living bacteria (approximately 20 nodules/larva for living bacteria vs. approximately 80 nodules/larva in insects challenged with heat-killed bacteria). The nodule-inhibiting action of living X. nematophilus was due to a factor that was present in the organic, but not aqueous, fraction of the bacterial cultural medium. The nodule-inhibiting factor in the organic fraction was labile to heat treatments. The immunodepressive influence of the factor in the organic fraction was reversed by treating challenged hornworms with arachidonic acid. The factor also depressed nodulation reactions to challenge with the plant pathogenic bacteria, Pseudomonas putida and Ralstonia solanacearum. These findings indicate that one or more factors from X. nematophilus depress nodulation reactions in tobacco hornworms by inhibiting eicosanoid biosynthesis.     

Simultaneous action of environmental factors on Candida utilis growth in a chemostat culture

The effect of carbon dioxide and carbon-containing metabolites on the growth of Candida utilis was studied under the conditions of phosphate limitation. The limiting factor and the hydrocarbonate form of CO2 were shown to act simultaneously on the yeast growth, decreasing its rate. The threshold concentration of the limiting factor remained unchanged. Carbon-containing metabolites produced a similar action on the chemostat yeast culture. The factors limiting the rate of the yeast growth did not switch over.     

Kinetics of batch single cell protein production from rice polishings with Candida utilis in continuously aerated tank reactors

Single cell protein was produced from the defatted rice polishings by fermentation with Candida utilis in an aerated 14-L fermentor to optimize bioprocess variables. Maximum values of specific growth rate coefficient (mu, h(-1)), cell mass yield (Y(X/S), g/g) and cell mass productivity (g/Lh) were 0.31, 0.65, and 1.24, respectively under optimized conditions of aeration rate (1 v.v(-1) m(-1)), dissolved oxygen (50%), corn steep liquor (5%), temperature (35 degrees C), and substrate concentration (90 g rice polishings/L) in yeast salt medium (pH 6.0). The kinetic parameters for 50-L fermentor under same conditions were 0.33 h(-1), 0.66 g/g, 1.33 g/Lh, 2.25 g/Lh, 1.23 g/Lh, 0.45 g/g substrate and 0.20 g/g cell h for mu, Y(X/S), Q(X), Q(S), Q(CP), Y(TP/S), and q(CP), respectively and were significantly higher than their respective values reported on C. utilis in batch culture studies. This biomass protein contained 23.6%, 32.75%, 11.50%, 12.95%, 10.5%, and 0.275% true protein, crude protein, crude fiber, ash, cellulose and RNA content respectively. This implied that the fermentation process could be up scaled to manufacture animal feed. Gross metabolizable energy content of dried SCP was 29,711 kcal/kg and indicated that the SCP could serve both as energy as well as a protein source. Yeast can replace expensive feed ingredients currently being incorporated in poultry feed and can reduce cost of poultry ration by 0.33 US dollars-0.51 US dollars/100 kg bag and improve the economics of feed production in our country.     

Comparative Study of Surface-Active Properties and Antimicrobial Activities of Disaccharide Monoesters

The objective of this research was to determine the effect of sugar or fatty acid in sugar ester compounds on the surface-active properties and antimicrobial activities of these compounds. Disaccharides of medium-chain fatty acid monoesters were synthesized through transesterifications by immobilized lipase (Lipozyme TLIM) to yield nine monoesters for subsequent study. Their antimicrobial activities were investigated using three pathogenic microorganisms: Staphylococcus aureus, Escherichia coli O157:H7 and Candida albicans. Their surface-active properties including air–water surface tension, critical micelle concentration, and foaming and emulsion power and stability were also studied. The results showed that all of the tested monoesters were more effective against Staphylococcus aureus (Gram-positive bacterium) than against Escherichia coli O157:H7 (Gram-negative bacterium). The results demonstrated that the carbon chain length was the most important factor influencing the surface properties, whereas degree of esterification and hydrophilic groups showed little effect.     

响应面法优化产朊假丝酵母培养及干燥工艺

本研究旨在优化产朊假丝酵母液体培养参数及干燥保护剂配方,提高产朊假丝酵母液体培养数量,制备出高复苏率的活性干酵母。以装液量、转速、温度和培养时间为影响因素,设计单因素试验,并采用Box-Behnken试验设计及响应面分析法优化产朊假丝酵母液体培养方案。在此条件下培养酵母,以L-谷氨酸钠、乳糖、脱脂奶粉为影响因素进行单因素保护试验,并采用响应面分析法优化干燥保护剂的组合。结果表明,产朊假丝酵母最佳培养条件为装液量45 mL/250 m L,转速200 r/min,温度30℃,培养时间24 h。在此条件下,培养的产朊假丝酵母数量可以达到9.34×10~8CFU/mL;最佳保护剂组合1%L-谷氨酸钠、12%脱脂奶粉、6%乳糖,经干燥后,产朊假丝酵母复苏率达到81.9%。此时活菌数7.65×10~8CFU/g,比未加保护剂组提高了3.83倍。     

Adaptation to temperature stress by Vibrio fischeri facilitates this microbe's symbiosis with the Hawaiian bobtail squid (Euprymna scolopes)

For microorganisms cycling between free‐living and host‐associated stages, where reproduction occurs in both of these lifestyles, an interesting inquiry is whether adaptation to stress during the free‐living stage can impact microbial fitness in the host. To address this topic, the mutualism between the Hawaiian bobtail squid (Euprymna scolopes) and the marine bioluminescent bacterium Vibrio fischeri was utilized. Using microbial experimental evolution, V. fischeri was selected to low (8°C), high (34°C), and fluctuating temperature stress (8°C/34°C) for 2000 generations. The temperatures 8°C and 34°C were the lower and upper growth limits, respectively. V. fischeri was also selected to benign temperatures (21°C and 28°C) for 2000 generations, which served as controls. V. fischeri demonstrated significant adaptation to low, high, and fluctuating temperature stress. V. fischeri did not display significant adaptation to the benign temperatures. Adaptation to stressful temperatures facilitated V. fischeri’s ability to colonize the squid host relative to the ancestral lines. Bioluminescence levels also increased. Evolution to benign temperatures did not manifest these results. In summary, microbial adaptation to stress during the free‐living stage can promote coevolution between hosts and microorganisms.     

Blood coagulation induced by Bothrops atrox venom: identification and properties of a factor X activator

The coagulating activity of Bothrops atrox venom was investigated in vitro with purified fibrinogen, with normal plasma and with plasma deficient in various factors of the coagulation cascade. This study indicated that Bothrops atrox venom possesses a thrombin-like action caused by one or several serine proteases sensitive to DFP treatment, but that its clotting action is in fact mainly due to components insensitive to DFP which activate prothrombin and factor X (Stuart factor). We partially purified the DFP insensitive activator of factor X from Bothrops atrox venom and found it to be a protein of molecular weight 77,000. Analysis of the purified fraction by electrophoresis on polyacrylamide gel in the presence of SDS showed that it is mainly composed of one heavy polypeptide chain (65,000) and one light doublet (12 - 13,000). This activator is calcium-dependent and catalyzes in vitro the conversion of purified factor X into factor X alpha. By its molecular properties, it resembles the factor X activator from Vipera russellii venom rather than physiological activators.     

Method for studying the adaptation of Escherichia coli to the acidification of the medium

Apparatus permitting to regulate the growth rate of turbidostat culture of microorganisms has been worked out. The process of adaptation of culture microorganisms to the medium acidity has been investigated by the stabilization of growth rate method. The correlation coefficients between adaptation time and changes of acid concentration in medium have been determined. The effect of aeration and density of microorganisms on the parameters of adaptation process has been considered. The conclusion concerning prospects of the growth rate stabilization method application for studying the regularities of culture microorganisms adaptation to the action of the stress has been made.     

Eicosanoids rescue Spodoptera exigua infected with Xenorhabdus nematophilus, the symbiotic bacteria to the entomopathogenic nematode Steinernema carpocapsae

Xenorhabdus nematophilus is a pathogenic bacterium causing insect haemolymph septicemia, which leads to host insect death. To address the fundamental mechanisms underlying this haemolymph septicemia, or the immunodepressive response of the host insects following bacterial infection, we tested a hypothesis that the insect immune-mediating eicosanoid pathway is blocked by inhibitory action of the bacterium. Haemocoelic injection of the bacteria into the fifth instar larvae of Spodoptera exigua reduced the total number of living haemocytes with postinjection time and resulted in host death in 16 h at 25 degrees C. The lethal efficacy, described by the median lethal bacterial dose (LD(50)), was estimated as 33 colony-forming units per fifth instar larva of S. exigua. The lethal effect of the bacteria on the infected larvae decreased significantly with the addition of exogenous arachidonic acid (10 μg), a precursor of eicosanoids. In comparison, injections of dexamethasone (10 μg), a specific inhibitor of phospholipase A(2), and other eicosanoid biosynthesis inhibitors elevated significantly the bacterial pathogenicity. Live X. nematophilus induced the infected larvae to form less nodules than did the heat-killed bacteria, but the addition of arachidonic acid increased the number of nodules formed significantly in response to live bacterial injection. The treatment with dexamethasone and other inhibitors, however, decreased the nodule formation after injection of heat-killed bacteria. These results indicate that eicosanoids play a role in the immune response of S. exigua, and suggest strongly that X. nematophilus inhibits its eicosanoid pathway, which then results in immunodepressive haemolymph septicemia.     

微生物对低温极端环境适应性的研究进展

嗜冷微生物是地球寒冷环境中最主要的生物类群,并且是驱动全球生物地球化学循环的关键环节。嗜冷微生物在适应策略上显示出应对多种极端环境因素的巨大潜力,研究其适应和进化机制有助于更好地理解微生物与环境之间相互作用过程,并有效利用极端环境微生物资源。近年来,随着分子生物学和基因组学技术的高速发展,对微生物适应寒冷环境的机制及嗜冷微生物在指示气候变化和工农业应用方面均有一系列的突破。在此,本文将从基因组的GC含量、蛋白质稳定性、转录翻译调控、细胞膜流动性、渗透压调节、抗氧化损失和基因组适应性进化等方面总结当前在微生物适应低温环境机制上所取得的进展,并展望低温环境微生物在指示气候变化和工农业应用中的前景。     

Production of volatile derivatives of metal(loid)s by microflora involved in anaerobic digestion of sewage sludge

Gases released from anaerobic wastewater treatment facilities contain considerable amounts of volatile methyl and hydride derivatives of metals and metalloids, such as arsine (AsH(3)), monomethylarsine, dimethylarsine, trimethylarsine, trimethylbismuth (TMBi), elemental mercury (Hg(0)), trimethylstibine, dimethyltellurium, and tetramethyltin. Most of these compounds could be shown to be produced by pure cultures of microorganisms which are representatives of the anaerobic sewage sludge microflora, i.e., methanogenic archaea (Methanobacterium formicicum, Methanosarcina barkeri, Methanobacterium thermoautotrophicum), sulfate-reducing bacteria (Desulfovibrio vulgaris, D. gigas), and a peptolytic bacterium (Clostridium collagenovorans). Additionally, dimethylselenium and dimethyldiselenium could be detected in the headspace of most of the pure cultures. This is the first report of the production of TMBi, stibine, monomethylstibine, and dimethylstibine by a pure culture of M. formicicum.     

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.     

Transfructosylation of thiol group by beta-fructofuranosidases

Beta-fructofuranosidase fructosylated not only the hydroxyl group but also the thiol group of 2-mercaptoethanol in a transfer reaction using sucrose as a donor substrate. The enzymes from Candida utilis and Saccharomyces cerevisiae (bakers' yeast) were effective catalysts for the thio-fructofuranosylation. The thio-fructosylation product was isolated by activated carbon chromatography and its structure was confirmed by Fab-mass spectrometry and NMR spectroscopy. The thio-fructofuranoside was synthesized effectively at around 3.0 M for the acceptor concentration. The product increased with the sucrose concentration at least up to 1.9 M. O-Fructofuranoside was simultaneously synthesized at an early stage of the reaction, although it was hydrolyzed on further incubation. On the contrary, the thio-fructofuranoside accumulated efficiently after synthesis, indicating it was very stable against the hydrolytic action of the beta-fructofranosidase.     

Method of culturing microorganisms at constant concentrations of the nutrient components

A method for batch cultivation of microorganisms in a flow medium is described, characterized by slight changes in concentrations of medium components in time and by the absence of products of vital activity of microorganisms in the fermentation medium. The conditions are achieved due to application of a fermentation installation with a microfiltrative membrane that separates the cells of cultivated microorganisms from the culture fluid and due to increasing the flow rate to a value at which the inlet and outlet concentrations of the medium components are almost equal. The cells of cultivated microorganisms under such conditions remain in the fermentation medium volume. The system was called "Ekostat". If the process is performed in "Ekostat" system, a positive deviation from the logarithmic law is observed for the growth rate of the yeast Candida utilis VSB-651 on ethanol cultivation.     

High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1

The low ethanol tolerance of thermophilic anaerobic bacteria, generally less than 2% (v/v) ethanol, is one of the main limiting factors for their potential use for second generation fuel ethanol production. In this work, the tolerance of thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 to exogenously added ethanol was studied in a continuous immobilized reactor system at a growth temperature of 70°C. Ethanol tolerance was evaluated based on inhibition of fermentative performance e.g. inhibition of substrate conversion. At the highest ethanol concentration tested (8.3% v/v), the strain was able to convert 42% of the xylose initially present, indicating that this ethanol concentration is not the upper limit tolerated by the strain. Long-term strain adaptation to high ethanol concentrations (6–8.3%) resulted in an improvement of xylose conversion by 25% at an ethanol concentration of 5% v/v, which is the concentration required in practice for economically efficient product recovery. For all ethanol concentrations tested, relatively high and stable ethanol yields (0.40–0.42 g/g) were seen. The strain demonstrated a remarkable ethanol tolerance, which is the second highest displayed by thermophilic anaerobic bacteria known to the authors. This appears to be the first study of the ethanol tolerance of these microorganisms in a continuous immobilized reactor system.     

Structural analysis of the elongation factor G protein from the low-temperature-adapted bacterium Arthrobacter globiformis SI55

The first structural analysis of elongation factor G (EF-G) from a cold-adapted bacterium is presented. EF-G is an essential protein involved in the elongation process during protein synthesis and is therefore thought to play a crucial role in the low-temperature adaptation of cold-adapted microorganisms. To define its importance, the EF-G gene (fus) from the psychrotolerant bacterium Arthrobacter globiformis SI55 was cloned and sequenced. The deduced primary structure of the elongation factor is composed of 700 amino acids with a predicted molecular mass of 77.4 kDa. A three-dimensional model of the protein was constructed based on the known crystal structures of structurally homologous proteins. Structural features that might potentially be important for activity and flexibility at low temperature were deduced by comparisons with models of the EF-G proteins from the closely related mesophiles Micrococcus luteus and Mycobacterium tuberculosis. These features include a loss in the number of salt bridges in intradomain and interdomain positions, increased solvent interactions mediated by greater charge and polarity on domain surfaces, loop insertions, loss of proline residues in loop structures, and an increase of hydrophobicity in core regions. Specific changes have also been identified in the catalytic domain (G domain) and sites of potential ribosome interaction, which may directly affect guanosine triphosphate (GTP) hydrolysis and elongation rates at low temperature. Received: September 20, 1999 / Accepted: December 2, 1999     

Antibacterial metabolites of lactic acid bacteria: their diversity and properties

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.     

Extremophilic microorganisms: Biochemical adaptation and biotechnological application (review)

In this review, we analyzed modern data on the biochemical adaptation of microorganisms to living under extreme conditions. Special attention is given to analysis of adaptation responses of microorganisms under exposure to increased radiation at molecular and cellular levels. Data on the practical use of extremophiles as well as extremoenzymes, biologically active compounds, biopolymers, etc., synthesized by them are systematized.     

Forced dissociation of selectin-ligand complexes using steered molecular dynamics simulation

Selectin-ligand interactions are crucial to such biological processes as inflammatory cascade or tumor metastasis. How transient formation and dissociation of selectin-ligand bonds in blood flow are coupled to molecular conformation at atomic level, however, has not been well understood. In this study, steered molecular dynamics (SMD) simulations were used to elucidate the intramolecular and intermolecular conformational evolutions involved in forced dissociation of three selectin-ligand systems: the construct consisting of P-selectin lectin (Lec) and epidermal growth factor (EGF)-like domains (P-LE) interacting with synthesized sulfoglycopeptide or SGP-3, P-LE with sialyl Lewis X (sLe(X)), and E-LE with sLe(X). SMD simulations were based on newly built-up force field parameters including carbohydrate units and sulfated tyrosine(s) using an analogy approach. The simulations demonstrated that the complex dissociation was coupled to the molecular extension. While the intramolecular unraveling in P-LE-SGP-3 system mainly resulted from the destroy of the two anti-parallel beta sheets of EGF domain and the breakage of hydrogen-bond cluster at the Lec-EGF interface, the intermolecular dissociation was mainly determined by separation of fucose (FUC) from Ca2+ ion in all three systems. Conformational changes during forced dissociations depended on pulling velocities and forces, as well as on how the force was applied. This work provides an insight into better understanding of conformational changes and adhesive functionality of selectin-ligand interactions under external forces.