Tom B. Morrison

Chemotactic Signaling by an Escherichia coli CheA Mutant That Lacks the Binding Domain for Phosphoacceptor Partners

CheA is a multidomain histidine kinase for chemotaxis in Escherichia coli. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheAs signaling specificity, we constructed CheADeltaP2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheADeltaP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheADeltaP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheADeltaP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.

Massively parallel microfluidics platform for storage and ultra-high-throughput screening

We have developed a novel microarray technology for performing very large numbers of biochemical, chemical and cell-based nanoliter volume synthesis, storage and screening operations in a massively parallel manner. The Living Chip is an array of precisely machined through-holes retaining nanoliters of fluid by capillary action. Sample loading, washing and recovery are operations that can be performed manually or with simple automation. Mixing between co- registered through-holes is achieved by stacking two or more precision aligned arrays and optical assay read-outs are in parallel with a CCD imaging system. An automated picker system transfers hits into lower density microtiter plates for further analysis. We will present result demonstrating massively parallel implementation of both homogeneous and inhomogeneous fluidic and cell-based assay systems and applications of the chip for drug compound library storage and management.