Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics

Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large‐scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co‐evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.     

Microtubule Depolymerization Inhibits Ethanol-Induced Enhancement of GABAA Responses in Stably Transfected Cells

Abstract: We studied whether microtubule organization is important for actions of ethanol on GABA_A ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk⁻) cells stably transfected with GABA_A receptor α₁β₁γ_2L subunits. The microtubule-disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol-induced enhancement of muscimol-stimulated chloride uptake. β-Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicine did not alter the potentiating actions of flunitrazepam or pentobarbital on muscimol-stimulated chloride uptake. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol-induced enhancement of GABA_A responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system.     

A novel and high-throughput approach to assess photosynthetic thermal tolerance of kelp using chlorophyll α fluorometry

Foundation seaweed species are experiencing widespread declines and localized extinctions due to increased instability of sea surface temperature. Characterizing temperature thresholds are useful for predicting patterns of change and identifying species most vulnerable to extremes. Existing methods for characterizing seaweed thermal tolerance produce diverse metrics and are often time-consuming, making comparisons between species and techniques difficult, hindering insight into global patterns of change. Using three kelp species, we adapted a high-throughput method – previously used in terrestrial plant thermal biology – for use on kelps. This method employs temperature-dependent fluorescence (T–F₀) curves under heating or cooling regimes to determine the critical temperature (T_crit) of photosystem II (PSII), i.e., the breakpoint between slow and fast rise fluorescence response to changing temperature, enabling rapid assays of photosynthetic thermal tolerance using a standardized metric. This method enables characterization of T_crit for up to 48 samples per two-hour assay, demonstrating the capacity of T–F₀ curves for high-throughput assays of thermal tolerance. Temperature-dependent fluorescence curves and their derived metric, T_crit, may offer a timely and powerful new method for the field of phycology, enabling characterization and comparison of photosynthetic thermal tolerance of seaweeds across many populations, species, and biomes.     

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. II. The beef heart mitochondrial system.

1. Beef heart mitochondrial ATPase, in both the membrane-bound and isolated form, contains tightly bound ATP and ADP. Each mol of ATPase contains about 2.2 mol ATP and 1.3 mol ADP. 2. In the absence of ATPase activity, these nucleotides exchange only slowly with nucleotides in solution. The exchange rate is increased during coupled ATPase activity, but not when the ATPase is uncoupled. 3. Oligomycin and dicyclohexylcarbodiimide inhibit exchange of the bound nucleotides, as does the ATPase inhibitor protein, although in each case some residual exchange occurs. Aurovertin, although inhibiting phosphorylation, does not inhibit the exchange. This is discussed in terms of the reversibility of these inhibitors. 4. The stimulation of exchange seen during coupled ATPase activity requires energisation of the ATPase molecule. Using the exchange reaction as a probe of energisation, it is deduced that energy can be transferred between different ATPase molecules. 5. It is proposed that coupled ATPase activity and phosphorylation in submitochondrial particles involve the tight nucleotide binding sites and the (weak) ATPase site, while uncoupled ATPase activity involves only the weak site.     

The receptors responsible for heat production in brown adipose tissue in the young rabbit

It is known that adrenergic agonists stimulate thermogenesis in the brown fat of the young rabbit but the receptors responsible for mediating the response have not been identified. The infusion of either noradrenaline or isoproterenol (1-2 micrograms . kg-1 X min-1) produced an increase in subcutaneous temperature (0.93 +/- 0.15 and 1.22 +/- 0.10 degrees C, respectively over the interscapular brown fat. At low doses (0.4 microgram . kg-1 X min-1) only isoproterenol was effective. The thermogenic response to isoproterenol was blocked by atenolol, a beta 1-adrenergic antagonist. Neither salbutamol or terbutaline, both beta 2-agonists, produced a temperature increase. Collectively, these data suggest that stimulation of beta 1-adrenoceptor is primarily responsible for the thermogenic activity of brown fat in the rabbit. However, it was found that 53% of the increase in temperature could be blocked by prazosin, an alpha 1-antagonist. Phentolamine was not effective as a blocker. Although a maximal brown fat thermogenic response can be achieved by stimulating the beta-adrenoceptors, the alpha-adrenoceptors appears to play at least an auxiliary role in young rabbit.     

A Dual Role for Corneal Dendritic Cells in Herpes Simplex Keratitis: Local Suppression of Corneal Damage and Promotion of Systemic Viral Dissemination

The cornea is the shield to the foreign world and thus, a primary site for peripheral infections. However, transparency and vision are incompatible with inflammation and scarring that may result from infections. Thus, the cornea is required to perform a delicate balance between fighting infections and preserving vision. To date, little is known about the specific role of antigen-presenting cells in viral keratitis. In this study, utilizing an established murine model of primary acute herpes simplex virus (HSV)-1 keratitis, we demonstrate that primary HSV keratitis results in increased conventional dendritic cells (cDCs) and macrophages within 24 hours after infection. Local depletion of cDCs in CD11c-DTR mice by subconjuntival diphtheria toxin injections, led to increased viral proliferation, and influx of inflammatory cells, resulting in increased scarring and clinical keratitis. In addition, while HSV infection resulted in significant corneal nerve destruction, local depletion of cDCs resulted in a much more severe loss of corneal nerves. Further, local cDC depletion resulted in decreased corneal nerve infection, and subsequently decreased and delayed systemic viral transmission in the trigeminal ganglion and draining lymph node, resulting in decreased mortality of mice. In contrast, sham depletion or depletion of macrophages through local injection of clodronate liposomes had neither a significant impact on the cornea, nor an effect on systemic viral transmission. In conclusion, we demonstrate that corneal cDCs may play a primary role in local corneal defense during viral keratitis and preserve vision, at the cost of inducing systemic viral dissemination, leading to increased mortality.