Circadian rhythms are important biological signals that have been found in almost all major groups of life from bacteria to man. Yet, it remains unclear if any members of the second major prokaryotic domain of life, the Archaea, also possess a biological clock. From an evolutionary perspective, the study of the Archaea is of great relevance, as their origin and relationship to the eukaryotes and bacteria thus far remains unresolved. Interestingly, DNA sequence information has revealed the presence of circadian gene homologs, known as kaiC, throughout a number of diverse archaeal genomes. KaiC is a major driver of the cyanobacterial circadian clock that acts to regulate rhythmic gene expression and to control the timing of cell division. To date, experimental evidence has not been provided to explain a functional role for any of these archaeal kaiC homologs. This project focuses on examining both the genetic regulation of four kaiC homologs found in the genome of the model system, the obligate halophilic, or salt-loving, archaeon Haloferax volcanii, as well as performing targeted gene knockouts to ascertain their true function. Evidence suggests that these four genes are regulated by diurnal cycles of light and darkness. Specific objectives for this research have been designed to define and characterize what role, if any, these circadian rhythm genes are playing in the Archaea. The following questions are addressed: How important is each of the four H. volcanii circadian-like genes in contributing to diurnal light sensing?; Are these genes inter-dependent for proper function? A multi-faceted experimental approach will involve classical genetics and microbiology, molecular biology, and protein biochemistry. These experiments will give a more complete picture regarding the nature of the ubiquitous kaiC homologs found among the Archaea. These results will provide the necessary first steps for better understanding if Archaea do indeed have some type of circadian clock, cyanobacterial-like, or otherwise. Alternatively, if the circadian clock hypothesis is dispelled following these experiments, the results will nevertheless be important in improved understanding of the role played by the haloarchaeal kaiC homologs, and, by association, the kaiC homologs found among the Archaea.
Broader impacts All of the proposed research will take place at Rider University, a small, liberal arts school that does not have graduate programs in the sciences. Thus, undergraduate involvement in this work is imperative for the timely completion of the proposed experiments. The PI is committed to the training of undergraduates in bench research, public speaking, and scientific writing. Greater than 70% of students mentored in the Bidle lab over the last nine years (n=40) have been female and 30% were underrepresented minorities (African-, Caribbean-, Latin-, or Arab-Americans). Nearly half of these 40 students have been accepted into graduate (both Ph.D. and Master's level) or professional (Medical, Nursing, Dental, Veterinary) programs. Students mentored in the Bidle lab have been the recipients of national research awards in microbiology, have been listed as authors on peer-reviewed journal publications and present their data at national scientific meetings each year. These students are trained in fundamental techniques covering a host of disciplines including genetics, molecular biology, bioinformatics, and microbiology. Hands-on, experiential learning is fostered in the Bidle lab, leading to the graduation of well-trained, next-generation life scientists.
Intellectual Merit: This project revolved around the study of circadian rhythms, important biological signals that have been found in almost all major groups of life ranging from bacteria to man. Our laboratory was interested in learning if members from the second major prokaryotic domain of life, the Archaea, also possess a biological clock. Our research revealed that Archaea encode genes, called "kai" (the Japanese word for cycle), that appear similar to those found in bacteria that exhibit circadian rhythms. Kai genes are the major driver of the cyanobacterial circadian clock, acting to regulate rhythmic gene expression and to control the timing of cell division. Prior to our research, experimental evidence had not been provided to explain a functional role for any of these archaeal kai gene homologs. To study archaeal kai-like genes, we used the model system Haloferax volcanii, an obligate halophile (or salt-lover) naturally found in the Dead Sea. As a result of our work, we have presented compelling evidence that demonstrates that H. volcanii gene expression is regulated by diurnal cycles of light and darkness. Given the ubiquity of kai homologs throughout the domain Archaea, we wished to further dissect how and why these genes may be present in this domain of life. Our research focused on addressing the following questions: How important are each of the four H. volcanii kai genes in contributing to diurnal light sensing?; Are each of these genes inter-dependent on each other for proper function? To address our objectives, we employed a multi-faceted experimental approach involving classical genetics and microbiology, molecular biology, and protein biochemistry. We examined the expression of each of the four kai genes in response to 12 h light/12 h dark cycles (LD 12:12) in H. volcanii during steady-state growth. Our data reveal that there is an approximately two to sixteen-fold increase in kai gene expression when cells are shifted from light to constant darkness, and this pattern of gene expression oscillates with the light conditions in a rhythmic manner. Targeted single- and double-gene knockouts in the H. volcanii kai genes result in disruption of light-dependent, rhythmic gene expression, although it does not lead to any significant effect on growth under these conditions. Restoration of light-dependent, rhythmic gene expression was demonstrated by introducing a wild-type, or fully functioning, copy of individual kai genes into knockout strains. These results are noteworthy as this is the first attempt to characterize the transcriptional expression and regulation of the ubiquitous kai homologs found among archaeal genomes. Broader Impacts: All of the proposed research took place at Rider University, a small, liberal arts school that does not have graduate programs in the sciences. All of the experiments conducted in the Bidle laboratory are performed by students majoring in Biology, Behavioral Neuroscience, Biochemistry and Environmental Science. The PI has been committed to the training of undergraduates in bench research, public speaking, and scientific writing for over 15 years, with many of her research students advancing into Ph.D. graduate programs. Over the past four years of this funding, following graduation, five students directly working on this project have been accepted to University of California, San Francisco, Rutgers University, Harvard University, University of Pennsylvania and University of Maryland. Much of this can be directly attributed to the fact that these students have had outstanding training in foundational laboratory bench skills, due in part to this funding. Greater than 70% of students mentored in the Bidle lab over the last 15 years (n=60) have been female and 30% were underrepresented minorities (African-, Caribbean-, Latin-, or Arab-Americans). Students mentored in the Bidle lab have been the recipients of national research awards, have been listed as authors on peer-reviewed journal publications and present their data at national scientific meetings each year. These students are trained in fundamental techniques covering a host of disciplines including genetics, molecular biology, bioinformatics, and microbiology.
