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.

School leaders as both colonized and colonizers: understanding professional identity in an era of no child left behind

This study positioned the federal No Child Left Behind (NCLB) Act of 2002 as a reified colonizing entity, inscribing its hegemonic authority upon the professional identity and work of school principals within their school communities of practice. Pressure on educators and students intensifies each year as the benchmark for Adequate Yearly Progress under the NCLB policy is raised, resulting in standards-based reform, scripted curriculum and pedagogy, absence of elective subjects, and a general lack of autonomy critical to the work of teachers as they approach each unique class and student (Crocco & Costigan, 2007; Mabry & Margolis, 2006). Emphasis on high stakes standardized testing as the indicator for student achievement (Popham, 2005) affects educators’ professional identity through dramatic pedagological and structural changes in schools (Day, Flores, & Viana, 2007). These dramatic changes to the ways our nation conducts schooling must be understood and thought about critically from school leaders’ perspectives as their professional identity is influenced by large scale NCLB school reform. The author explored the impact No Child Left Behind reform had on the professional identity of fourteen, veteran Illinois principals leading in urban, small urban, suburban, and rural middle and elementary schools. Qualitative data were collected during semi-structured interviews and focus groups and analyzed using a dual theoretical framework of postcolonial and identity theories. Postcolonial theory provided a lens from which the author applied a metaphor of colonization to principals’ experiences as colonized-colonizers in a time of school reform. Principal interview data illustrated many examples of NCLB as a colonizing authority having a significant impact on the professional identity of school leaders. This framework was used to interpret data in a unique and alternative way and contributed to the need to better understand the ways school leaders respond to district-level, state-level, and national-level accountability policies (Sloan, 2000). Identity theory situated principals as professionals shaped by the communities of practice in which they lead. Principals’ professional identity has become more data-driven as a result of NCLB and their role as instructional leaders has intensified. The data showed that NCLB has changed the work and professional identity of principals in terms of use of data, classroom instruction, Response to Intervention, and staffing changes. Although NCLB defines success in terms of meeting or exceeding the benchmark for Adequate Yearly Progress, principals’ view AYP as only one measurement of their success. The need to meet the benchmark for AYP is a present reality that necessitates school-wide attention to reading and math achievement. At this time, principals leading in affluent, somewhat homogeneous schools typically experience less pressure and more power under NCLB and are more often labeled “successful” school communities. In contrast, principals leading in schools with more heterogeneity experience more pressure and lack of power under NCLB and are more often labeled “failing” school communities. Implications from this study for practitioners and policymakers include a need to reexamine the intents and outcomes of the policy for all school communities, especially in terms of power and voice. Recommendations for policy reform include moving to a growth model with multi-year assessments that make sense for individual students rather than one standardized test score as the measure for achievement. Overall, the study reveals enhancements and constraints NCLB policy has caused in a variety of school contexts, which have affected the professional identity of school leaders.

Single particle motion of polymeric, colloidal, and biological materials

Small particles and their dynamics are of widespread interest due both to their unique properties and their ubiquity. Here, we investigate several classes of small particles: colloids, polymers, and liposomes. All these particles, due to their size on the order of microns, exhibit significant similarity in that they are large enough to be visualized in microscopes, but small enough to be significantly influenced by thermal (or Brownian) motion. Further, similar optical microscopy and experimental techniques are commonly employed to investigate all these particles. In this work, we develop single particle tracking techniques, which allow thorough characterization of individual particle dynamics, observing many behaviors which would be overlooked by methods which time or ensemble average. The various particle systems are also similar in that frequently, the signal-to-noise ratio represented a significant concern. In many cases, development of image analysis and particle tracking methods optimized to low signal-to-noise was critical to performing experimental observations. The simplest particles studied, in terms of their interaction potentials, were chemically homogeneous (though optically anisotropic) hard-sphere colloids. Using these spheres, we explored the comparatively underdeveloped conjunction of translation and rotation and particle hydrodynamics. Developing off this, the dynamics of clusters of spherical colloids were investigated, exploring how shape anisotropy influences the translation and rotation respectively. Transitioning away from uniform hard-sphere potentials, the interactions of amphiphilic colloidal particles were explored, observing the effects of hydrophilic and hydrophobic interactions upon pattern assembly and inter-particle dynamics. Interaction potentials were altered in a different fashion by working with suspensions of liposomes, which, while homogeneous, introduce the possibility of deformation. Even further degrees of freedom were introduced by observing the interaction of particles and then polymers within polymer suspensions or along lipid tubules. Throughout, while examination of the trajectories revealed that while by some measures, the averaged behaviors accorded with expectation, often closer examination made possible by single particle tracking revealed novel and unexpected phenomena.