This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific Aims: 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. Tudor has demonstrated that the composition of the bdellocyst is unique to the W strain and different from other cyst-forming bacteria such as Rhodospirillum centenum, but Tudor could not identify its exact components. 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 the bdellocyst is unique to Bdellovibrio bacteriovorus W). Investigating the regulation of cyst development in Bdellovibrio bacteriovorus W using proteomic and genetic techniques is expected to complement the results obtained by the sequencing of the genome of strain W. 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 formation 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 Bdellovibrio bacteriovorus W. The rationale of undertaking a proteomic approach to address bdellocyst 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. The rationale behind taking the genetic approach to identify the genes that control bdellocyst formation in Bdellovibrio bacteriovorus W is that Bdellovibrio bacteriovorus DNA is amenable to transposon mutagenesis by electroporation, and thus transposon mutants defective in bdellocyst formation can be identified. The availability of the Escherichia coli Keio library that is comprised of open reading frame deletions of non-essential genes makes screening for host genes involved in encystment possible. We plan to test our central hypothesis by identifying the genes that regulate cyst development in Bdellovibrio W (bdellocyst) by pursuing the following two specific aims specific aims. 1. Specific aim one: 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. To test this hypothesis the following experimental approach will be undertaken. 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). Time course studies of protein expression changes upon bdellocyst germination and encystment will be conducted using two-dimensional protein electrophoresis as well as PF2D. After performing two-dimensional separation of proteins at each time point, protein profiles will be compared, and differentially expressed proteins will be analyzed and characterized further by mass spectrophotometry. 2. Specific aim two: Identification of genes that control bdellocyst Development in Bdellovibrio bacteriovorus W by means of transposon mutagenesis. We hypothesize that the genes that regulate bdellocyst formation in Bdellovibrio bacteriovorus W are unique.To test this hypothesis, the following experimental approach will be undertaken. Gene knockout experiments: The purpose behind these series of experiments is to identify the genes that control bdellocyst development of Bdellovibrio bacteriovorus W. Bdellovibrio bacteriovorus W is able to form bdellocysts i.e. switch to an encystment mode when it infects its host upon starvation. Bdellocysts are resistant to desiccation, they are able to germinate once the desiccated bdellocysts are exposed to an environment conducive to germination. It is expected that the genes that control bdellocyst development are unique. In order to identify the genes that control bdellocyst development, a Tn5 transposon library of vegetative Bdellovibrio bacteriovorus W cells will be created by electroporation. W clones from the transposon mutant library will be screened for their ability to form bdellocysts by infecting their host cells under conditions conducive to encystment followed by desiccating the cultures. The mutants that fail to germinate after desiccation will be the W bdellocysts mutants. The DNA that flanks the bdellocyst mutant transposon insertion sites will be sequenced and the W genes that control encystment identified. In order to investigate the role of host bacteria in the process of encystment in Bdellovibrio bacteriovorus W, a Keio library that comprises Escherichia coli non-essential genes will be screened for genes that prevent bdellocyst formation.