Population Dynamics of Chlamydomonas sajao and Its Influence on Soil Aggregate Stabilization in the Field

Chlamydomonas sajao, a single-celled, eucaryotic microalga, was inoculated onto replicated field plots cropped to corn at two rates (5 × 10¹¹ and 5 × 10⁷ log-phase cells ha⁻¹) to assess colonization, reproduction, and persistence, changes in soil carbohydrate content, and wet stability of 0.92- to 1.68-mm-sized aggregates from the surface 2- to 3-mm soil veneer. The most-probable-number technique and extraction and fluorometric quantification of chlorophyll were used as indices of microalgal abundance. Cell numbers ranged up to 6.9 × 10⁶ g of soil⁻¹ by most probable number and were significantly greater on high-rate than on low-rate and control plots over a 10-week period. Chlorophyll content and most probable number were positively correlated (r = 0.64) for the high-rate plots but not for low-rate plots or the controls. Increased wet aggregate stability measurements (33 to 77%) for the high-rate plots during the growing season were significantly greater than for low-rate and control plots, which were not different from each other. The work leads support to the hypothesis that mass-cultured palmelloid microalgae are a feasible means for conditioning soil biologically.     

Improved heterologous erythromycin A production through expression plasmid re‐design

The production of complex compounds from technically convenient microorganisms is an emerging route to the chemical diversity found in the surrounding environment. In this study, the antibiotic compound erythromycin A is produced from Escherichia coli as an alternative to native production through the soil bacterium Saccharopolyspora erythraea. By doing so, there is an opportunity to apply and refine engineering strategies for the manipulation of the erythromycin biosynthetic pathway and for the overproduction of this and other complex natural compounds. Previously, E. coli‐derived production was enabled by the introduction of the entire erythromycin pathway (20 genes total) using separately selectable expression plasmids which demonstrated negative effects on final biosynthesis through metabolic burden and plasmid instability. In this study, improvements to final production were made by altering the design of the expression plasmids needed for biosynthetic pathway introduction. Specifically, the total number of genes and plasmids was pruned to reduce both metabolic burden and plasmid instability. Further, a comparison was conducted between species‐specific (E. coli vs. S. coelicolor) protein chaperonins. Results indicate improvements in growth and plasmid retention metrics. The newly designed expression platform also increased erythromycin A production levels 5‐fold. In conclusion, the steps outlined in this report were designed to upgrade the E. coli erythromycin A production system, led to improved final compound titers, and suggest additional forms of pathway engineering to further improve results from heterologous production attempts. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:862–869, 2013     

REH2C Helicase and GRBC Subcomplexes May Base Pair through mRNA and Small Guide RNA in Kinetoplastid Editosomes

Mitochondrial mRNAs in Trypanosoma brucei undergo extensive insertion and deletion of uridylates that are catalyzed by the RNA editing core complex (RECC) and directed by hundreds of small guide RNAs (gRNAs) that base pair with mRNA. RECC is largely RNA-free, and accessory mitochondrial RNA-binding complex 1 (MRB1) variants serve as scaffolds for the assembly of mRNA-gRNA hybrids and RECC. However, the molecular steps that create higher-order holoenzymes (“editosomes”) are unknown. Previously, we identified an RNA editing helicase 2-associated subcomplex (REH2C) and showed that REH2 binds RNA. Here we showed that REH2C is an mRNA-associated ribonucleoprotein (mRNP) subcomplex with editing substrates, intermediates, and products. We isolated this mRNP from mitochondria lacking gRNA-bound RNP (gRNP) subcomplexes and identified REH2-associated cofactors 1 and 2 (^H2F1 and ^H2F2). ^H2F1 is an octa-zinc finger protein required for mRNP-gRNP docking, pre-mRNA and RECC loading, and RNP formation with a short synthetic RNA duplex. REH2 and other eukaryotic DEAH/RHA-type helicases share a conserved regulatory C-terminal domain cluster that includes an oligonucleotide-binding fold. Recombinant REH2 and ^H2F1 constructs associate in a purified complex in vitro. We propose a model of stepwise editosome assembly that entails controlled docking of mRNP and gRNP modules via specific base pairing between their respective mRNA and gRNA cargo and regulatory REH2 and ^H2F1 subunits of the novel mRNP that may control specificity checkpoints in the editing pathway.     

Resolving UV Bioactinometric Results with Intensity and Time Distributions

A UV reactor with an annular design, a total liquid volume of 460[emsp4 ]ml, and outfitted with a single lamp with 1690[emsp4 ]mW of germicidal power was tested. Coliphage MS2 was used as a bioactinometer to measure the UV dose at a flow rate of 56.7[emsp4 ]ml/sec in water with a very low absorbance. The Beers Law coefficient was A₁₀0.003. The measured dose (MS2 bioactinometry) was 35.2±1.1[emsp4 ]mW-sec/cm².A retention time distribution was generated with a dye tracer study. The reactor was modeled as if flow was confined to ten equal volume paths existing as concentric rings around the lamp. The UV intensity along each path (ith intensity) was calculated to generate a simulated distribution of UV intensity in the reactor. The retention time distribution was subdivided to estimate the retention time associated with each decile jth time) of the total flow.Seven methods of associating the ith intensity with the jth retention time were used to produce simulated dose distributions for the reactor. The average UV dose for each distribution was calculated as the average of the products of I and t (AP protocol) and by the apparent survival (AS protocol), in which the predicted survival along each path was averaged to back-calculate dose from the reference batch inactivation curve. The average dose predicted assuming that time and intensity were independent was 51.5[emsp4 ]mW-sec/cm² based on the arithmetic average (AP protocol). Using the apparent survival method, the predicted dose for the independent distribution (I independent of t) was 36.4[emsp4 ]mW-sec/cm². Three methods of developing dependent structure between time and intensity were tested. In the best possible case for stratified flow (I negatively correlated with t) the calculated (AS) intensity was 46.3[emsp4 ]mW-sec/cm^2. In the worst case for stratified flow (I positively correlated with t) the AS intensity was 32.0[emsp4 ]mW-sec/cm². In a rational case where flows were assumed to be distributed parabolically (low flow at the wall and at the lamp) produced an AS intensity of 37.7[emsp4 ]mW-sec/cm². When either time or intensity was averaged, while the other variable was allowed to keep its distribution, the (AS) dose (time averaged 43.3[emsp4 ]mW-sec/cm², intensity averaged 41.0[emsp4 ]mW-sec/cm²), yielded a poor prediction compared to the measured value.The errors associated with averaging time, intensity, or both, far outweigh the errors associated with choosing a rational distribution or an independent distribution of time and intensity in the prediction. This observation is generally true whenever an organism is exposed to UV light in a flow through reactor such that the range of doses is within the portion of the inactivation curve exhibiting strong exponential decay.