Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen

SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity

JTRF Volume 56 No. 3, Fall 2017A Framework to Analyze the Feasibilityof Vehicle Miles Traveled Fees to Financea Sustainable Transportation System

This paper presents a framework to analyze vehicle miles travelled (VMT) fees as an alternative to fi nance maintenance, rehabilitation, and new construction transportation projects. The VMT feasibility framework addresses major factors related to public acceptance, revenues, technology, type of contract, government policies, enforcement, administration, and invoicing. We argue that our suggested VMT fee policy is an equitable usage-based system since in our analysis, VMT fees are differentiated by vehicle axles and emissions. In turn, VMT charges will also motivate fl eet owners to renew vehicles or switch to alternative transportation modes such as mass transit, walking, and biking. An example based on data from the state of Texas illustrates some of the potential revenues and benefi ts associated with a VMT fee policy