Conditional,Temperature-Induced Proteolytic Regulation of Cyanobacterial RNA Helicase Expression

Conditional proteolysis is a crucial process regulating the abundance of key regulatory proteins associated with the cell cycle, differentiation pathways, or cellular response to abiotic stress in eukaryotic and prokaryotic organisms. We provide evidence that conditional proteolysis is involved in the rapid and dramatic reduction in abundance of the cyanobacterial RNA helicase, CrhR, in response to a temperature upshift from 20 to 30°C. The proteolytic activity is not a general protein degradation response, since proteolysis is only present and/or functional in cells grown at 30°C and is only transiently active at 30°C. Degradation is also autoregulatory, since the CrhR proteolytic target is required for activation of the degradation machinery. This suggests that an autoregulatory feedback loop exists in which the target of the proteolytic machinery, CrhR, is required for activation of the system. Inhibition of translation revealed that only elongation is required for induction of the temperature-regulated proteolysis, suggesting that translation of an activating factor was already initiated at 20°C. The results indicate that Synechocystis responds to a temperature shift via two independent pathways: a CrhR-independent sensing and signal transduction pathway that regulates induction of crhR expression at low temperature and a CrhR-dependent conditional proteolytic pathway at elevated temperature. The data link the potential for CrhR RNA helicase alteration of RNA secondary structure with the autoregulatory induction of conditional proteolysis in the response of Synechocystis to temperature upshift.     

Effect of substrate and IPTG concentrations on the burden to growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> on glycerol due to the expression of Lac proteins

Expression of proteins unneeded for growth diverts cellular resources from making necessary protein and leads to a reduction in the growth rate of an organism. This reduction in growth rate is termed as cost. Cost plays an important role in determining the selected expression of a protein in a particular environment. Characterization of cost is important in biotechnology industries where microorganisms are used to produce foreign proteins. We have used the lactose system in Escherichia coli to quantify the cost of growth on glycerol in the presence of isopropyl-β-d-thiogalactopyranoside (IPTG), an inducer of the lactose system. The effect of the concentration of the carbon source, glycerol, and the inducer of Lac enzymes, IPTG, is studied. The results show that the cost is dependent on the glycerol concentration with a decreasing trend with increasing concentration of glycerol. Also as expected, the cost increases and saturates at a higher concentration of IPTG. The studies also demonstrate that the cost is higher in early exponential phase relative to late exponential phase during the growth as has been reported in the literature. Hill equation fit yielded a typical Monod-type expression for growth on glycerol with and without IPTG. An apparent half-saturation constant was defined which was used to characterize the burden on growth due to protein expression.     

SSB, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes

Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.     

The Interrelationship between Promoter Strength,Gene Expression,and Growth Rate

In exponentially growing bacteria, expression of heterologous protein impedes cellular growth rates. Quantitative understanding of the relationship between expression and growth rate will advance our ability to forward engineer bacteria, important for metabolic engineering and synthetic biology applications. Recently, a work described a scaling model based on optimal allocation of ribosomes for protein translation. This model quantitatively predicts a linear relationship between microbial growth rate and heterologous protein expression with no free parameters. With the aim of validating this model, we have rigorously quantified the fitness cost of gene expression by using a library of synthetic constitutive promoters to drive expression of two separate proteins (eGFP and amiE) in E. coli in different strains and growth media. In all cases, we demonstrate that the fitness cost is consistent with the previous findings. We expand upon the previous theory by introducing a simple promoter activity model to quantitatively predict how basal promoter strength relates to growth rate and protein expression. We then estimate the amount of protein expression needed to support high flux through a heterologous metabolic pathway and predict the sizable fitness cost associated with enzyme production. This work has broad implications across applied biological sciences because it allows for prediction of the interplay between promoter strength, protein expression, and the resulting cost to microbial growth rates.     

Economic assessment of plant cell culture for the production of ajmalicine

Cost estimates have been prepared for commercial-scale production of ajmalicine-rich Catnaranthus roseus biomass using plant cell culture. At the current state of the technology the cost would be approximately $7.30/lb dry biomass ($3215/kg ajmalicine). Naturally-grown C. roseus roots have a 50% lower ajmalicine concentration but would cost only ca. $0.70/lb ($619/kg ajmalicine). The principal reason for the high cost of the plant cell culture route is not the slow specific growth rate (0.35 day(-1)), but rather the slow specific product accumulation rate (0.26 mg/g day). This rate will have to be increased by a factor of 40 to make the process competitive.