Intellectual Merit: Bacteria are everywhere on earth because they are masters at adapting to environmental changes. They sense their environment through specialized receptor proteins, such as the cAMP receptor protein (CRP) of Escherichia coli. This CRP protein has become the model for understanding transcriptional regulation in research and teaching. With many bacterial genomes sequenced in recent years, many CRP homologs have been discovered throughout the eubacteria. By analogy to E. coli CRP, these CRP homologs are predicted to be DNA binding proteins whose DNA binding activity is modulated by ligand binding. The main goal of this project is to apply the accumulated knowledge of the E. coli CRP to discover functions of these diverse CRP homologs. To identify the gene targets of the CRP homologs, the CRP homologs first will be mutated to introduce changes that should make them constitutively active; then the mutants will be used in SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments to identify their cognate high affinity target DNA sequences. A second strategy will identify ligands that influence activity of several CRP homologs and explore which amino acid residues are important for ligand recognition.
Broader Impacts: The scientific goals of this project will be achieved using a format of integrated research and class teaching. A substantial portion of this research will be conducted by students within two lab-intensive courses: a molecular biology lab (~20 graduate students over two years, course offered once a year) and a microbiology lab (~30 undergraduate students over two years, course offered once a year). This hypothesis-driven project will give the students an excellent inquiry-based learning opportunity to experience how science is done and to consider science as a career option. Also, because approximately 40% of the students at California State University, Fresno are from underrepresented ethnic groups, this research project will have a significant impact on minority students.
This RIG project applied the principal investigator’s (PI’s) expertise on the Escherichia coli cAMP receptor protein (CRP), a model bacterial transcription factor, to several other proteins similar to CRP (termed CRP homologs) from various bacteria to understand their diverse functions. More than 20 CRP homologs and their chimeric variants were cloned and characterized, with research focused on two essential functions: ligand binding and DNA binding. Significant progress was made in elucidating those functional aspects of the CRP homologs, which resulted in a publication. The publication demonstrated that the in vitro cAMP response of Vfr, a Pseudomonas aeruginosa CRP homolog, is in good agreement with the physiological (virulent) role of the protein in vivo. In particular, two functionally important differences between CRP and Vfr were noted: (1) Vfr has a significantly higher affinity for cAMP than CRP, which might explain unidirectional complementation between the two proteins. (2) Vfr is activated by both cAMP and cGMP, while CRP is specific to cAMP. It was also shown that a single amino acid residue, Thr133, is the key residue for the distinctive properties of Vfr. Furthermore, a remarkable class of Vfr variants was identified that have completely reversed the regulatory logic of the protein: they are active in DNA binding without cAMP and are strongly inhibited by cAMP. This body of work provides further testable hypotheses on the ligand sensing and response of Vfr. The activities of this project involved a total of 70 students: 13 research students (undergraduate and Master’s students) were trained through the PI’s research lab and 67 class students (from microbiology and molecular biology courses) conducted hypothesis-driven research in classroom settings. The hypothesis-driven projects provided an excellent inquiry-based learning opportunity for the class students. These activities of students led to 12 poster and 2 oral presentations at local, national and international conferences. Additionally, the activities provided the early stage PI with an excellent professional development opportunity regarding both research and teaching, thereby making the PI a better educator at California State University, Fresno. The data acquired from this project will help the PI carry out more grant-assisted research in the future. The project also served as a basis for developing a new course, "Functional Genomics" and for improving other courses taught by the PI, including "Microbial Physiology" and "Nucleic Acid Technology Lab." In conclusion, the project generated research/educational outcomes fulfilling the mission of the NSF RIG program.
