Investigation of the roles of CheD in the Bacillus subtilis chemotactic signal transduction

Abstract The two-component based chemotaxis signal transduction system allows flagellated bacteria to sense their surrounding chemical environment and move towards more favorable conditions. The attractant signals can be sensed by transmembrane chemoreceptors, and then transmitted to the histidine kinase CheA. Once activated, CheA interacts with the response regulator CheY through phosphorelay, which causes a change in the rotation of the flagella. The direction of flagella rotation determines whether a cell swims straight or just tumbles. Cells also need adaptation to respond to a change in chemical concentrations, and return to their prestimulated level. Adaptation in the B. subtilis chemotaxis system is achieved by three coordinated systems: the methylation system, the CheC/CheD/CheY-p system and the CheV system. CheD, the previously identified receptor deamidase, was shown to be critical to the ability of B. subtilis to perform chemotaxis and is the main focus of this study. This study started from characterization of the enzymatic mechanism of CheD. Results showed that CheD deamidase uses a cysteine hydrolase mechanism. The catalytic triad consisting of Cys33-His50-Thr27, and Ser27 is essential for receptor recognition and binding. In addition, in this study CheC was found to inhibit CheD’s deamidase activity. Through mutant screening, Phe102 on CheD was found to be the essential site to interact with CheC. Furthermore, the CheD/CheC interaction is necessary for the robust chemotaxis in vivo as demonstrated by the cheD (F102E) mutant, which lacks the ability to swim on swarm plates. Despite its deamidase activity, we hypothesized that CheD’s main role is its involvement in the CheD-CheC-CheY-p negative feedback pathway during adaptation. In particular, CheD is likely to help stabilize the transient kinase-activating state through binding to receptors. When CheY-p level is increased, CheC-CheY-p complex may attract CheD away from receptors. In this study, CheC-CheD binding kinetics with CheY or CheYp presence was successfully obtained by a series of SPR experiments. The increased affinity of CheD for CheC in presence of CheYp but not CheY makes likely the hypothesis that CheC-CheD-CheY interact as part of a negative feedback pathway during adaptation. Last, the interaction between CheD and chemoreceptor McpC was studied in order to better understand the role of CheD in adaptation. Results showed that Q304 and Q305 on McpC are essential to recruit CheD. Additionally, the reduced levels of CheD in mcpC (Q304A) or (Q305A) mutants suggested that the dynamic interaction between CheD and receptors is vital to maintain the normal CheD level. These findings suggest more complicated roles of CheD than its previously identified function as a receptor deamidase, and will lead to a clearer picture of the coordination of the three adaptational systems in the B. subtilis chemotactic sensory transduction system.

Organizational responses to technological discontinuities: the case of the American College of Radiology (ACR)

A long history of organizational research has shown that organizations are affected significantly by changes in technology. Scholars have given particular attention to the effects of so-called disruptive or discontinuous technological changes. Studies have repeatedly shown that established, incumbent organizations tend to suffer deep performance declines (and even complete demise) in the face of such changes, and researchers have devoted much attention to identifying the organizational conditions and processes that are responsible for this persistent and widespread pattern of adaptation failure. This dissertation, which examines the response of the American College of Radiology (ACR) to the emergence of nuclear magnetic resonance imaging technology (NMR), aims to contribute to this well-established research tradition in three distinct and important ways. First, it focuses on a fundamentally different type of organization, a professional association, rather than the technology producers examined in most prior research. Although technologies are well known to be embedded in “communities” that include technology producers, suppliers, customers, governmental entities, professional societies, and other entities, most prior research has focused on the responses and ultimate fate of producers alone. Little if any research has explored the responses of professional organizations in particular. Second, the study employs a sophisticated process methodology that identifies the individual events that make up the organization’s response to technological change, as well as the overall sequence through which these events unfold. This process approach contrasts sharply with the variance models used in most previous studies and offers the promise of developing knowledge about how adaptation ultimately unfolds (or fails to). Finally, the project also contributes significantly through its exploration of an apparently successful case of adaptation to technological change. Though nuclear magnetic resonance imaging posed a serious threat to the ACR and its members, this threat appears to have been successfully managed and overcome. Although the unique nature of the organization and the technology under study place some important limits on the generalizablity of this research, its findings nonetheless provide some important basic insights about the process through which social organizations can successfully adapt to discontinuous technological changes. These insights, which may also be of substantial relevance to technology producer organizations, will also be elaborated.