This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.A.
Specific Aims :This project has only been active 4 months and progress will reflect efforts to date. Bdellovibrio bacteriovorus is a gram-negative bacterium that preys on a variety of gram-negative bacteria; some of them are the human pathogens Pseudomonas aerunginosa, Salmonella typhimurium and E. coli O157:H7. Bdellovibrio bacteriovorus is characterized by a biphasic life cycle: a flagellated motile non-dividing 'attack phase' and a 'growth phase' where Bdellovibrio multiplies inside the host cell. When Bdellovibrio forms the requisite number of progeny inside the host cell, it bursts from the host cell. Bdellovibrio bacteriovorus strain W is the only strain among the Bdellovibrios that is able to form a cyst (bdellocyst) inside its prey under adverse environmental conditions such as starvation. There is a scant body of research that tackles the genetic and molecular aspects of bdellocyst development as well as its uniqueness. (Until the present time, nothing is known about the genetic and molecular aspects of cyst development or why bdellocyst is unique to Bdellovibrio bacteriovorus W).. Investigating the regulation of cyst development in Bdellovibrio bacteriovorus W using proteomic techniques is expected to complement the results obtained by the sequencing project in Dr. Iandolo's laboratory. Identification of the genes that regulate cyst development is expected to provide us with an understanding of the unique phenomenon of bdellocyst development and evolution in Bdellovibrio bacteriovorus W.The ultimate long-term goal is to explore the evolutionary significance of bdellocyst acquisition by Bdellovibrio bacteriovorus W by bdellocyst mutant analysis. The objective of this application is to identify and characterize the genes that regulate bdellocyst development. The central hypothesis is that encystment in Bdellovibrio bacteriovorus W as well as its unique composition are additional survival adaptations that serve to control prey lysis much more stringently. The rationale behind investigating the control of cyst development in Bdellovibrio bacteriovorus W is that the presence and the composition of the bdellocyst are unique to bacteriovorus W. The rationale of undertaking a proteomic approach to address cyst development is three-fold. First, transcriptional changes do not necessarily result in translational changes. Second, the core facility at OUHSC has recently acquired the high resolution ProteomeLab' PF2D which greatly increases the probability of success of taking a proteomic approach to study regulation of bdellocyst development. Third, the sequencing of Bdellovibrio bacteriovorus W is almost completed. We plan to test our hypothesis by identifying the genes that regulate cyst development in Bdellovibrio W (bdellocyst) by pursuing the following specific aim. Identification of differentially expressed proteins in bdellocyst germination by two-dimensional electrophoresis and mass spectrophotometry: We hypothesize that upon the process of encystment and germination in Bdellovibrio w some proteins are expected to be upregulated and others to be downregulated. The approach that I will be undertaking to study bdellocyst development in Bdellovibrio bacteriovorus strain W is as follows. Bdellovibrio bacteriovorus W will be maintained in Rhodospirillum rubrum prey bacteria. Proteome experiments: The purpose behind this series of experiments is to tackle the regulation of the process of encystment and the process of germination in Bdellovibrio bacteriovorus strain W using a proteomic approach. It is expected that there will be a change in the protein profiles during the course of bdellocyst germination and of encystment (bdellocyst formation) in strain W: some proteins will be induced (up-regulated) whereas others will disappear (down-regulated). Differentially expressed proteins obtained from the proteome experiments will be subject to Tryptic digestion. Tryptic maps will be generated from the digests of each protein chosen by mass spectrophotometry. The Tryptic maps will be queried against proteomic databases such as MS-FIT, and thus the genes involved in bdellocyst development could be identified. The proposed work is innovative as the discovery and characterization of the genes that regulate cyst development using high resolution PF2D proteomics is expected to provide us with a better understanding about the uniqueness of bdellocyst presence and composition in Bdellovibrio bacteriovorus W. It is also expected to shed light on the link between encystment in Bdellovibrio bacteriovorus W and the control of lysis of prey cells. Once the genes involved in bdellocyst development are identified, molecular and genetic studies such as differential gene expression and creation of mutants can be conducted.
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