Revised enzyme synthesis rate expression in cybernetic models of bacterial growth

A revised enzyme synthesis rate expression for cybernetic models of bacterial growth is presented. The rate expression, which is comprised of inducible and constitutive contributions, provides for a basal enzyme level that is necessary to predict certain types of commonly observed continuous culture transients. The response of a continuous culture to a step change in feed stream composition is simulated using both the old and new formulations, and the ramifications for the "matching-law" formulation are discussed.     

Volatilization of mercury by immobilized mercury-resistant bacterial cells

Highly toxic mercury compounds may come into the environment through the use of mercury compounds as disinfectants for hospital and household purposes, Hg catalyst in industries, burning of coal and petroleum products, mercury-based pesticides and fungicides used in agriculture, and seed dressings. Toxic effects of mercury can be counteracted by microbial cells through the enzymes mercuric reductase and organomercurial lyase. Immobilized mercury-resistant bacterial cells of Azotobacter chroococcum could effectively volatilize mercury from mercury-containing buffer and detoxify mercury compounds. Moreover, the efficiency of mercury volatilization was much greater than with the native cells, as immobilized cells can be reused. Immobilized cells continuously volatilized mercury from mercury-containing buffer after four consecutive 24 h cycles. The storage stability of immobilized cells was much better than that of the native cells.     

Crystal Structure of a Bioactive Pactamycin Analog Bound to the 30S Ribosomal Subunit

Biosynthetically and chemically derived analogs of the antibiotic pactamycin and de-6-methylsalicylyl (MSA)-pactamycin have attracted recent interest as potential antiprotozoal and antitumor drugs. Here, we report a 3.1-Å crystal structure of de-6-MSA-pactamycin bound to its target site on the Thermus thermophilus 30S ribosomal subunit. Although de-6-MSA-pactamycin lacks the MSA moiety, it shares the same binding site as pactamycin and induces a displacement of nucleic acid template bound at the E-site of the 30S. The structure highlights unique interactions between this pactamycin analog and the ribosome, which paves the way for therapeutic development of related compounds.     

Probing functional diversity in pactamycin toward antibiotic,antitumor, and antiprotozoal activity

A total of eight new analogs of pactamycin were prepared and tested alongside pactamycin and three of its natural congeners for antibacterial, anticancer, and antiprotozoal activities. The present study highlights the effects of changing the urea and aniline groups especially with regard to anticancer and antiprotozoal activities.     

Screening of metal uptake by plant colonizers growing on abandoned copper mine in Kapunda,South Australia

Systematic site survey for sample collection and analysis was conducted at a derelict copper (Cu) mine at Kapunda, South Australia. Cu concentrations in the soils at this former mine ranged from 65–10107 mg kg^?1. The pH and EC varied widely in the 3.9–8.4 and 152–7311 µS ranges, respectively. Nine plant species growing over the copper mine site were selected to screen for metal uptake to determine their suitability for phytoremediation. The Australian native tree species Eucalyptus camaldulensis indicated enrichment factor (EF) of 2.17, 1.89, and 1.30 for Cu, Zn, and Pb, respectively, suggesting that this species of tree can accumulate these metals to some degree. The stress-resistant exotic olive, Olea europaea exhibited EF of ≤ 0.01 for Cu, Cd, and Pb, and 0.29 for Zn, which is characteristic of an excluder plant. Acacia pycnantha, the Australian pioneer legume species with EF 0.03, 0.80, 0.32, and 0.01 for Cu, Zn, Cd, and Pb, respectively, emerged as another strong metal excluder and consequently as an ideal metal stabilizer.     

Metabolic engineering from a cybernetic perspective: aspartate family of amino acids

Using the modular cybernetic framework developed by Varner and Ramkrishna (Varner and Ramkrishna; 1998a, b) a cybernetic model is formulated that describes the time evolution of the aspartate family of amino acids in Corynebacterium lactofermentum ATCC 21799. The network model formulation is employed in the role of a diagnostic tool for the overproduction of threonine. More precisely, having determined a parameter set that describes the time evolution of a base strain (lysine producer), the model predicted response to genetic perturbations, designed to enhance the level of threonine, are simulated using an appropriately modified cybernetic model and compared with the experimental results of Stephanopoulos and Sinskey (Colón et al., 1995a, Appl. Environ. Microbiol. 61, 74-78) for identical genetic perturbations. It is found that the model predicted response to enzymatic over-expression in the aspartate pathway agrees, for the most part, with experimental observations within the experimental error bounds. This result lends credence to the hypothesis that cybernetic models can be employed to predict the local response of a metabolic network to genetic perturbation, thereby, affording cognizance of the potential pitfalls of a particular genetic alteration strategy a priori.     

Unveiling steady-state multiplicity in hybridoma cultures: the cybernetic approach

Mammalian cells grown in suspension produce waste metabolites such as lactate, alanine, and ammonia, which reduce the yield of cell mass and the desired product on the nutrients supplied. Previous studies (Cruz et al., 1999; Europa et al., 2000; Follstad et al., 1999) have shown that the cells can be made to alter their metabolism by starving them on their nutrients in continuous cultures at low dilution rates or starting the culture as a fed-batch. This leads to multiple steady states in continuous reactors, with some states being more favorable than others. Mathematical models that take into account the metabolic regulation that leads to these multiple steady states are invaluable tools for bioreactor control. In this article we present a cybernetic modeling strategy in which Metabolic Flux Analysis (MFA) is used to guide the cybernetic formulation. The hybridoma model presented as a result of this strategy considers the partially substitutable, partially complementary nature of glucose and glutamine. The choice of competitions within the network is guided by MFA and the model is successful in explaining the three multiple steady states observed. The cybernetic model though identified for the hybridoma experiments of Hu and others (Europa et al., 2000) seem generally applicable to mammalian systems as it captures the pathways that are common to mammalian cells grown in suspension. The model presented here could be used for start-up strategies for continuous reactors and model-based feedback control for maintaining high productivity of the reactor.     

Engineering surface hydrophobicity improves activity of Bacillus thermocatenulatus lipase 2 enzyme

Bacillus thermocatenulatus lipase 2 (BTL2) is a promising industrial enzyme used in biodiesel production. Although BTL2 has high thermostability and good resistance to organic solvents, the activity of BTL2 is suboptimal for industrial processes. To improve BTL2 activity, we engineered BTL2 lipase by modulating hydrophobicity of its lid domain. Through site‐directed mutagenesis, we constructed three mutants, namely Y225F+S232A, S232A+T236V and Q185L, to cover all uncharged hydrophilic amino acids within the lid domain. Activities of these mutants were characterized. Our findings suggest that one mutant (Y225F+S232A) showed ～35% activity increase in catalyzing heterogeneous hydrolytic reactions relevant for industrial applications. A mathematical framework was established to account for different molecular events that contribute to the observed apparent catalytic activities. Increases in hydrophobicity of lid domains were associated with increased interfacial adsorption of lipases and lower molecular enzymatic activities. The measured apparent activities of lipases include contributions from both events. Lid hydrophobicity can thus result in different changes in lipase activities depending on the mutation site. Our work demonstrates the feasibility of increasing BTL2 activity by modulating the hydrophobicity of lid domains and provides some guidelines for further improving BTL2 activity.     

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.