Intellectual Merit: The overall goal of this research is to understand the role of non-coding small RNAs (sRNAs) in controlling cellular processes in bacteria including survival in the environment. The bacterium, Vibrio cholerae, uses cell-cell communication, or quorum sensing (QS), to synchronously regulate expression of four Qrr RNAs (quorum regulatory RNAs) in response to the density of the bacterial population. Recent studies demonstrated that the Qrr sRNAs, like other bacterial sRNAs, can regulate multiple target genes. Although V. cholerae is a transient human pathogen, it is a common inhabitant of the aquatic estuarine and marine ecosystems. This research project is designed to exploit the understanding of the model V. cholerae QS system to discover the role of the Qrr sRNAs in influencing microbial behaviors in diverse environmental settings. Specifically, this project will address the following aims. Aim 1: To identify genes controlled by the quorum sensing-regulated sRNAs. Aim 2: To define the molecular mechanism of sRNA regulation using genetic, biochemical, and computational methods. Aim 3: To assess the conservation of QS circuitry and function in V. cholerae strains isolated from diverse locations. Because sRNAs are involved in important biological functions in all domains of life, defining the mechanism by which these sRNAs act will have fundamental implications for understanding regulatory circuitry of all cellular systems. Moreover, bacterial cell-cell communication systems are widespread in environmentally, commercially, and clinically significant microbes. An understanding of how these signaling pathways function, and how they control bacterial survival in the environment can ultimately engender strategies to curtail world-wide problems of environmental, human, animal and plant health.
Broader Impacts: The applicant engages in teaching and service activities aimed at disseminating modern scientific discovery and fostering interactions between members of the academic community and broader society. Training of students includes teaching an undergraduate lecture course and a course for upper level undergraduate Biology majors and graduate students in Prokaryotic Molecular Genetics. Collaborations initiated with researchers within the United States and in India promote international scientific and cultural exchange. Dr. Hammer has also developed an outreach program to engage ethnically diverse urban K-12 students in stimulating hands-on scientific activities that will be shared with local educators who teach under-represented minority students in the metro-Atlanta area. He is a volunteer for science outreach events at Georgia Tech and in the surrounding community, an ad hoc reviewer for multiple journals. He is an active teacher and mentor currently of three undergraduate and two graduate students, and one postdoctoral researcher.
Intellectual Merit: The overall goal of this research study was to understand the role of non-coding small RNAs (sRNAs) in controlling cellular processes in bacteria. We stated that understanding the significance that sRNAs play in regulating cellular processes requires target identification, and definition of the molecular mechanisms governing the sRNA/mRNA base pairing interactions. The focus of this research was on the model organism Vibrio cholerae, which uses a cell-cell communication process, called quorum sensing (QS), to express four Qrr sRNAs (quorum regulatory RNAs) in response to the bacteria population density. From the work conducted in this study we published one paper in which we used in silico, in vivo and in vitro analyses to demonstrate that the Qrrs directly base pair with and repress translation of hapR mRNA. HapR is a transcription factor that coordinates expression of >100 genes including those for biofilm formation, protease secretion, DNA uptake and cholera toxin. We showed that Qrr pairing is essential for these processes to be properly executed. In addition, using the tools we developed, we also published a study detailing that the same sRNAs also directly base pair with and activate translation of vca0939 mRNA, which we showed encodes an enzyme for making an intracellular signal (c-di-GMP) that promotes biofilm formation. A study that continues in the lab will define an additional direct target gene of the Qrr sRNAs as well as the role of this mRNA target in V. cholerae. We published a methods paper detailing techniques used to document sRNA/mRNA pairing, and a review paper describing the broader role of sRNAs in V. cholerae. Finally, the observation that Qrr/hapR pairing regulates DNA uptake (transformation) led to an independent project that served as the basis of a new NSF proposal studying DNA uptake. Broader Impacts: The proposed research included service and teaching activities that engaged members of the academic community and the broader society in modern scientific discovery. In addition to teaching two courses (Prokaryotic Molecular Genetics and Regulatory RNAs); a substantial outreach program was developed to engage metro-Atlanta K-12 students in stimulating hands-on scientific activities. A fifth and seventh grade teacher joined the Hammer lab each summer in 2010-2012 to pilot activities that were then used in the public school classroom. Each summer Dr. Hammer and the teacher were assisted by one undergraduate student considering a career in K-12 teaching. In November 2012, Dr. Hammer and the seventh grade teacher presented a "Germs to Genes" workshop to over 80 participants at the National Science Teachers Association (NSTA) meeting in Atlanta. Hammer also volunteered for science outreach events on campus and in the community, was an ad hoc reviewer for multiple journals, and most importantly mentored 16 lab members: 9 undergraduates, 4 MS students, 2 PhD students that recently defended their PhDs and accepted postdoctoral research positions, and 1 postdoctoral researcher still in the laboratory. Teaching, mentoring and outreach activities continue.
