Design and Selection of a Synthetic Feedback Loop for Optimizing Biofuel Tolerance.

Feedback control allows cells to dynamically sense and respond to environmental changes. However, synthetic controller designs can be challenging because of implementation issues, such as determining optimal expression levels for circuit components within a feedback loop. Here, we addressed this by coupling rational design with selection to engineer a synthetic feedback circuit to optimize tolerance of Escherichia coli to the biojet fuel pinene. E. coli can be engineered to produce pinene, but it is toxic to cells. Efflux pumps, such as the AcrAB-TolC pump, can improve tolerance, but pump expression impacts growth. To address this, we used feedback to dynamically regulate pump expression in response to stress. We developed a library with thousands of synthetic circuit variants and subjected it to three types of pinene treatment (none, constant, and varying pinene). We were able to select for strains that were biofuel tolerant without a significant growth cost in the absence of biofuel. Using next-generation sequencing, we found common characteristics in the designs and identified controllers that dramatically improved biofuel tolerance.

Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis.

The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.