Time-Restricted Feeding Prevents Obesity and Metabolic Syndrome in Mice Lacking a Circadian Clock

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Activities of a Mechanosensitive Ion Channel in anE. Coli Mutant Lacking the Major Lipoprotein

Summary The activity of the mechanosensitive (MS) ion channels in membrane patches, excised fromE. coli spheroplasts, was analyzed using the patch-clamp technique. Outer membranes from a mutant lacking the major lipoprotein (Lpp) and its wildtype parent were examined. The MS-channel activities in the wild-type membrane rarely revealed substates at the time resolution used. These channels showed a stretch sensitivity indicated by the IISP (the suction for ane-fold increase in channel open probability) of 4.9 mm Hg suction. The MS-channel activities oflpp included a prominent substate and showed a weaker mechano-sensitivity with an 1/S _p of 10.0 mm Hg. Whereas small amphipaths (chlorpromazine, trinitrophenol) or a larger amphipath (lysolecithin) all activated the MS channel in the wild-type membrane under minimal suction, only the larger lysolecithin could activate the MS channel in thelpp membranes. After lysolecithin addition, thelpp membrane became more effective in transmitting the stretch force to the MS channel, as indicated by a steepening of the Boltzmann curve. We discuss one interpretation of these results, in which the major lipoprotein serves as a natural amphipath inserted in the inner monolayer and the loss of this natural amphipath makes the bilayer less able to transmit the gating force.     

Metal Resistance and Lithoautotrophy in the Extreme Thermoacidophile Metallosphaera sedula

Archaea such as Metallosphaera sedula are thermophilic lithoautotrophs that occupy unusually acidic and metal-rich environments. These traits are thought to underlie their industrial importance for bioleaching of base and precious metals. In this study, a genetic approach was taken to investigate the specific relationship between metal resistance and lithoautotrophy during biotransformation of the primary copper ore, chalcopyrite (CuFeS₂). In this study, a genetic system was developed for M. sedula to investigate parameters that limit bioleaching of chalcopyrite. The functional role of the M. sedula copRTA operon was demonstrated by cross-species complementation of a copper-sensitive Sulfolobus solfataricus copR mutant. Inactivation of the gene encoding the M. sedula copper efflux protein, copA, using targeted recombination compromised metal resistance and eliminated chalcopyrite bioleaching. In contrast, a spontaneous M. sedula mutant (CuR1) with elevated metal resistance transformed chalcopyrite at an accelerated rate without affecting chemoheterotrophic growth. Proteomic analysis of CuR1 identified pleiotropic changes, including altered abundance of transport proteins having AAA-ATPase motifs. Addition of the insoluble carbonate mineral witherite (BaCO₃) further stimulated chalcopyrite lithotrophy, indicating that carbon was a limiting factor. Since both mineral types were actively colonized, enhanced metal leaching may arise from the cooperative exchange of energy and carbon between surface-adhered populations. Genetic approaches provide a new means of improving the efficiency of metal bioleaching by enhancing the mechanistic understanding of thermophilic lithoautotrophy.     

Altered Lipid Droplet Dynamics in Hepatocytes Lacking Triacylglycerol Hydrolase Expression

Lipid droplets (LDs) form from the endoplasmic reticulum (ER) and grow in size by obtaining triacylglycerols (TG). Triacylglycerol hydrolase (TGH), a lipase residing in the ER, is involved in the mobilization of TG stored in LDs for the secretion of very-low-density lipoproteins. In this study, we investigated TGH-mediated changes in cytosolic LD dynamics. We have found that TGH deficiency resulted in decreased size and increased number of LDs in hepatocytes. Using fluorescent fatty acid analogues to trace LD formation, we observed that TGH deficiency did not affect the formation of nascent LDs on the ER. However, the rate of lipid transfer into preformed LDs was significantly slower in the absence of TGH. Absence of TGH expression resulted in increased levels of membrane diacylglycerol and augmented phospholipid synthesis, which may be responsible for the delayed lipid transfer. Therefore, altered maturation (growth) rather than nascent formation (de novo synthesis) may be responsible for the observed morphological changes of LDs in TGH-deficient hepatocytes.     

Oxygen Affects Gut Bacterial Colonization and Metabolic Activities in a Gnotobiotic Cockroach Model

The gut microbiota of termites and cockroaches represents complex metabolic networks of many diverse microbial populations. The distinct microenvironmental conditions within the gut and possible interactions among the microorganisms make it essential to investigate how far the metabolic properties of pure cultures reflect their activities in their natural environment. We established the cockroach Shelfordella lateralis as a gnotobiotic model and inoculated germfree nymphs with two bacterial strains isolated from the guts of conventional cockroaches. Fluorescence microscopy revealed that both strains specifically colonized the germfree hindgut. In diassociated cockroaches, the facultatively anaerobic strain EbSL (a new species of Enterobacteriaceae) always outnumbered the obligately anaerobic strain FuSL (a close relative of Fusobacterium varium), irrespective of the sequence of inoculation, which showed that precolonization by facultatively anaerobic bacteria does not necessarily favor colonization by obligate anaerobes. Comparison of the fermentation products of the cultures formed in vitro with those accumulated in situ indicated that the gut environment strongly affected the metabolic activities of both strains. The pure cultures formed the typical products of mixed-acid or butyrate fermentation, whereas the guts of gnotobiotic cockroaches accumulated mostly lactate and acetate. Similar shifts toward more-oxidized products were observed when the pure cultures were exposed to oxygen, which corroborated the strong effects of oxygen on the metabolic fluxes previously observed in termite guts. Oxygen microsensor profiles of the guts of germfree, gnotobiotic, and conventional cockroaches indicated that both gut tissue and microbiota contribute to oxygen consumption and suggest that the oxygen status influences the colonization success.     

Horizontal Transfer of Bacterial Symbionts: Heritability and Fitness Effects in a Novel Aphid Host

Members of several bacterial lineages are known only as symbionts of insects and move among hosts through maternal transmission. Such vertical transfer promotes strong fidelity within these associations, favoring the evolution of microbially mediated effects that improve host fitness. However, phylogenetic evidence indicates occasional horizontal transfer among different insect species, suggesting that some microbial symbionts retain a generalized ability to infect multiple hosts. Here we examine the abilities of three vertically transmitted bacteria from the Gammaproteobacteria to infect and spread within a novel host species, the pea aphid, Acyrthosiphon pisum. Using microinjection, we transferred symbionts from three species of natural aphid hosts into a common host background, comparing transmission efficiencies between novel symbionts and those naturally infecting A. pisum. We also examined the fitness effects of two novel symbionts to determine whether they should persist under natural selection acting at the host level. Our results reveal that these heritable bacteria vary in their capacities to utilize A. pisum as a host. One of three novel symbionts failed to undergo efficient maternal transmission in A. pisum, and one of the two efficiently transmitted bacteria depressed aphid growth rates. Although these findings reveal that negative fitness effects and low transmission efficiency can prevent the establishment of a new infection following horizontal transmission, they also indicate that some symbionts can overcome these obstacles, accounting for their widespread distributions across aphids and related insects.     

Growth and Metabolic Activities of Heat Treated Staphylococcus aureus

Staphylococcus aureus cells which had been heated at 50 or 60° were transferred to various growth media and intracellular ribonucleic acid (RNA), deoxyribonucleic acid (DNA) and amino acids were measured during the lag phase of growth. The duration of the lag phase depended on the temperature to which the cultures had been subjected, and was longest following storage at 60°. RNA synthesis occurred almost immediately on placing treated cells in a growth medium, but at a slower rate than with unheated cells. Variation in the composition of the metabolic pool of heated cells occurred during the early lag phase and may be as a result of damage to the cytoplasmic membrane with resulting loss of permeability control.     

Host Remodeling of the Gut Microbiome and Metabolic Changes during Pregnancy

Many of the immune and metabolic changes occurring during normal pregnancy also describe metabolic syndrome. Gut microbiota can cause symptoms of metabolic syndrome in nonpregnant hosts. Here, to explore their role in pregnancy, we characterized fecal bacteria of 91 pregnant women of varying prepregnancy BMIs and gestational diabetes status and their infants. Similarities between infant-mother microbiotas increased with children's age, and the infant microbiota was unaffected by mother's health status. Gut microbiota changed dramatically from first (T1) to third (T3) trimesters, with vast expansion of diversity between mothers, an overall increase in Proteobacteria and Actinobacteria, and reduced richness. T3 stool showed strongest signs of inflammation and energy loss; however, microbiome gene repertoires were constant between trimesters. When transferred to germ-free mice, T3 microbiota induced greater adiposity and insulin insensitivity compared to T1. Our findings indicate that host-microbial interactions that impact host metabolism can occur and may be beneficial in pregnancy.     

Metabolic Activities of Isolated Spores of Anabaena cylindrica

A method for the isolation of spores of Anabaena cylindricais described, and results concerning the metabolic activitiesof isolated spores are presented. Isolated spores fixed carbondioxide in the light at a lower rate and evolved carbon dioxidein the dark at a higher rate than the corresponding whole filaments.Nitrogen-fixing ability in isolated spores could be detectedneither with the isotopic method nor with the new acetylenetechnique. Isolated spores were able to germinate at the samerate as spores within the original cell population when transferredinto fresh culture medium.     

Expression in Escherichia coli of Native and Chimeric Phenolic Acid Decarboxylases with Modified Enzymatic Activities and Method for Screening Recombinant E. coli Strains Expressing These Enzymes

Four bacterial phenolic acid decarboxylases (PAD) from Lactobacillus plantarum, Pediococcus pentosaceus, Bacillus subtilis, and Bacillus pumilus were expressed in Escherichia coli, and their activities on p-coumaric, ferulic, and caffeic acids were compared. Although these four enzymes displayed 61% amino acid sequence identity, they exhibit different activities for ferulic and caffeic acid metabolism. To elucidate the domain(s) that determines these differences, chimeric PAD proteins were constructed and expressed in E. coli by exchanging their individual carboxy-terminal portions. Analysis of the chimeric enzyme activities suggests that the C-terminal region may be involved in determining PAD substrate specificity and catalytic capacity. In order to test phenolic acid toxicity, the levels of growth of recombinant E. coli displaying and not displaying PAD activity were compared on medium supplemented with different concentrations of phenolic acids and with differing pHs. Though these acids already have a slight inhibitory effect on E. coli, vinyl phenol derivatives, created during decarboxylation of phenolic acids, were much more inhibitory to the E. coli control strain. To take advantage of this property, a solid medium with the appropriate pH and phenolic acid concentration was developed; in this medium the recombinant E. coli strains expressing PAD activity form colonies approximately five times smaller than those formed by strains devoid of PAD activity.     

Division and Adaptation to Host Environment of Apicomplexan Parasites Depend on Apicoplast Lipid Metabolic Plasticity and Host Organelle Remodeling

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米曲霉外源表达系统研究进展

丝状真菌米曲霉是发酵工业的重要菌种,具有强大的蛋白分泌能力和较高的食品安全性,可作为表达外源蛋白的细胞工厂。近年来,米曲霉全基因组序列的测序完成和基于表达序列标签的基因组学研究,为深入研究米曲霉外源表达系统提供了条件。从基因组学进展、遗传转化体系等方面综述了米曲霉作为外源蛋白表达宿主的研究进展。针对米曲霉在外源蛋白表达中存在的瓶颈,提出构建蛋白酶缺陷株、使用强启动子、融合表达等策略,以提高外源蛋白的表达和产量。最后介绍了米曲霉表达系统的应用,利用米曲霉代谢工程菌生产工业用酶和次级代谢产品具有良好的前景。     

Denitrification in a Sand and Gravel Aquifer

Denitrification was assayed by the acetylene blockage technique in slurried core material obtained from a freshwater sand and gravel aquifer. The aquifer, which has been contaminated with treated sewage for more than 50 years, had a contaminant plume greater than 3.5-km long. Near the contaminant source, groundwater nitrate concentrations were greater than 1 mM, whereas 0.25 km downgradient the central portion of the contaminant plume was anoxic and contained no detectable nitrate. Samples were obtained along the longitudinal axis of the plume (0 to 0.25 km) at several depths from four sites. Denitrification was evident at in situ nitrate concentrations at all sites tested; rates ranged from 2.3 to 260 pmol of N₂O produced (g of wet sediment)⁻¹ h⁻¹. Rates were highest nearest the contaminant source and decreased with increasing distance downgradient. Denitrification was the predominant nitrate-reducing activity; no evidence was found for nitrate reduction to ammonium at any site. Denitrifying activity was carbon limited and not nitrate limited, except when the ambient nitrate level was less than the detection limit, in which case, even when amended with high concentrations of glucose and nitrate, the capacity to denitrify on a short-term basis was lacking. These results demonstrate that denitrification can occur in groundwater systems and, thereby, serve as a mechanism for nitrate removal from groundwater.     

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host''s oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.Download video file.^{(57M, mov)}     

Transfer and Expression of a Multiple Antibiotic Resistance Plasmid in Marine Bacteria

Conjugal transfer of a multiresistance plasmid from Pseudomonas fluorescens to halophilic and halotolerant bacteria was studied under in vitro and in situ conditions. Mating conducted in broth as well as on plates yielded a plasmid transfer frequency of as high as 10⁻³. Among these two, plate mating facilitated conjugal transfer of plasmid, because the cell-to-cell contact is more in plate mating. When P. fluorescens was incubated in seawater, the organism progressively lost its colony forming activity within 15 days. Microscopic examination revealed the presence of very short rods, indicating that the cells have become viable but nonculturable (VNC). Mating conducted in natural seawater without any added nutrients revealed that the conjugal transfer is influenced by the physical state of the donor and the recipients as well as the availability of nutrients. But a plasmid transfer frequency of 10⁻⁷ was obtained even after the donor cells have become VNC suggesting that the nonculturable state and nutrient deprived condition may not limit plasmid transfer. The results suggest that the terrestrial bacteria entering into the seawaters with antibiotic resistance plasmids may be responsible for the prevalence of resistance genes in the marine environment. Received: 4 May 1998 / Accepted: 18 June 1998