Molecular cloning and expression of the biodegradative threonine dehydratase gene ( tdc ) of Escherichia coli K12

The biodegradative threonine dehydratase gene ( tdc ) of Escherichia coli was cloned by isolating a dehydratase-negative mutant after Tn5 mutagenesis, cloning the tdc ::Tn5 DNA into pBR322 and then replacing the Tn5 element on the plasmid in vivo. Subcloning and nucleotide sequence data revealed two distinct procaryotic promoterlike elements each containing a potential CAP-binding site and AT-rich regions, and a Shine-Dalgarno sequence. One of these putative promoters, P 2 , was located immediately upstream from the tdc coding region, and a second, P 1 , was approximately 1 kilobase upstream from P 2 . Deletion of the potential CAP-binding site from P 1 prevented tdc gene expression. However, removal of P 2 and a large segment of the upstream DNA had no discernible effect on dehydratase synthesis. A 936-base pair open reading frame was found between P 1 and the tdc coding region, which produced a polypeptide of about 32 kilodaltons. The data suggest that P 1 , and not P 2 , is necessary for tdc gene expression, and that the DNA sequences coding for the 32 KD polypeptide and threonine dehydratase are part of a single transcriptional unit.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47562/1/438_2004_Article_BF00425676.pd

T7 RNA Polymerase Functions In Vitro without Clustering

Many nucleic acid polymerases function in clusters known as factories. We investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when active. Using ‘pulldowns’ and fluorescence correlation spectroscopy we find that elongation complexes do not interact in vitro with a Kd<1 µM. Chromosome conformation capture also reveals that genes located 100 kb apart on the E. coli chromosome do not associate more frequently when transcribed by T7 RNAP. We conclude that if clustering does occur in vivo, it must be driven by weak interactions, or mediated by a phage-encoded protein

Achieving Optimal Growth through Product Feedback Inhibition in Metabolism

Recent evidence suggests that the metabolism of some organisms, such as Escherichia coli, is remarkably efficient, producing close to the maximum amount of biomass per unit of nutrient consumed. This observation raises the question of what regulatory mechanisms enable such efficiency. Here, we propose that simple product-feedback inhibition by itself is capable of leading to such optimality. We analyze several representative metabolic modules—starting from a linear pathway and advancing to a bidirectional pathway and metabolic cycle, and finally to integration of two different nutrient inputs. In each case, our mathematical analysis shows that product-feedback inhibition is not only homeostatic but also, with appropriate feedback connections, can minimize futile cycling and optimize fluxes. However, the effectiveness of simple product-feedback inhibition comes at the cost of high levels of some metabolite pools, potentially associated with toxicity and osmotic imbalance. These large metabolite pool sizes can be restricted if feedback inhibition is ultrasensitive. Indeed, the multi-layer regulation of metabolism by control of enzyme expression, enzyme covalent modification, and allostery is expected to result in such ultrasensitive feedbacks. To experimentally test whether the qualitative predictions from our analysis of feedback inhibition apply to metabolic modules beyond linear pathways, we examine the case of nitrogen assimilation in E. coli, which involves both nutrient integration and a metabolic cycle. We find that the feedback regulation scheme suggested by our mathematical analysis closely aligns with the actual regulation of the network and is sufficient to explain much of the dynamical behavior of relevant metabolite pool sizes in nutrient-switching experiments

A Developmental Perspective on Community Service in Adolescence

A substantial number of U.S. adolescents currently participate in community service and there is increased national interest in service programs. This article assesses the assumption of developmental benefits to service participants by critically reviewing 44 empirical studies. It offers a theoretical framework for understanding the findings by connecting them to identity development and delineating three pertinent concepts: agency, social relatedness, and moral-political awareness. These concepts are applied to studies that investigate: ( 1) the characteristics and motivations of participants, ( 2) the effects of service, and ( 3) the process of service. The findings support the conclusion that service activities which provide opportunities for intense experiences and social interactions are often associated with prosocial development. The findings also point to the need for more studies focused on particular service programs and on relationships between service providers and those served