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<OL> <LI> Analyze the roles of the Cts and pVir encoded transformation systems during chick colonization. Using in vitro and in vivo transformation experiments, I will test the contributions to transformation of two different systems suggested to have roles in C. jejuni natural competence, the Cts and pVir systems. I will develop an in vivo co-culture model that will test mutations for colonization of the chick and transformation efficiencies in this model. <LI>Determine the cis- and trans-regulatory elements of the cts genes. Using an astA reporter fusion to the transformation system gene, ctsX, and transposon mutagenesis, I will identify regulatory elements of the cts genes. I will develop a selectable IVET system to study cts gene regulation in vivo. <LI>Role CtsX in natural transformation. I will identify proteins interacting with the membrane bound CtsX using crosslinking and co-immunoprecipitation experiments. I will construct mutant forms of CtsX to determine important domains for function of CtsX in natural transformation and interaction with other proteins identified during crosslinking and co-immunoprecipitation experiments.
NON-TECHNICAL SUMMARY: The bacterium Campylobacter jejuni is a leading cause of foodborne illness in humans. C. jejuni is commonly found in the chicken gastrointestinal tract, but does not cause disease. Infection by C. jejuni is typically caused by the ingestion of contaminated food and water sources. Campylobacter typically causes inflammatory diarrhea, but can manifest itself in more severe forms of disease. The spread of antibiotic resistance is an increasing concern in many bacterial species. Natural competence is one mechanism for the genetic transfer of both antibiotic resistance and virulence factors among bacteria. The goal of this project is to address genetic exchange and regulation, strain variation, and antibiotic resistance in the naturally competent bacterium Campylobacter jejuni. This project will identify factors important for the uptake and integration of extracellular DNA into the Campylobacter chromosome. A multiprotein system, called the Cts system, controls natural competence in C. jejuni, but the exact mechanism of action remains unclear at this time. Additionally, conditions in which natural competence is induced are not entirely understood. This proposal is designed to close gaps in our understanding of the mechanism and timing of Cts function.
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APPROACH: Traditional and emerging molecular biology techniques will be used to study the role of the Cts system in natural competence of Campylobacter. I will use the day-of-hatch chick model of Campylobacter infection to study natural transformation in vivo. Day-of-hatch chicks will be infected by oral gavage with streptomycin resistant DRH212 and 81-176 astA::cat. After seven days, I will sacrifice the chicks, harvest organs, and plate on non-selective media to obtain total amount of C. jejuni in the chick. Then, I will replica plate to screen for colonies that are chloramphenicol and streptomycin resistant, indicating the transformation of DNA between strains. In vitro transformation experiments are performed by adding purified DNA, carrying chloramphenicol resistance, to C. jejuni cells and then plating as described for the in vivo experiments. Traditional genetic approaches using reporter gene fusions and transposon mutagenesis will be used to identify regulators of the Cts system. I constructed an astA fusion to ctsX and will identify transcriptional regulators of the cts genes using this fusion. To do this, a transposon mutagenesis procedure previously described in this lab will be performed and colonies will be screened for changes in colony color on MH plates containing XS. Colonies with an increased level of blue color indicate an increase in ctsX expression and suggest that a repressor of cts expression has been disrupted. White colonies indicate that expression of ctsX has been lowered and suggest that an activator of cts has been disrupted. Once insertions altering the expression of cts have been isolated, the location of the insert will be identified by inverse PCR and semi-exponential cycle sequencing. The resulting DNA sequence will be compared to the C. jejuni 81-176 genome sequence. Finally, a resolvase-reporter system (selectable IVET) will be developed to study expression of cts genes during chicken colonization. This will be modified from an existing system used in E. coli to work in C. jejuni. This will also be applied more broadly to identify promoters activated by C. jejuni during growth in the chicken gastrointestinal tract. Co-immunoprecipation experiments will be carried out to identify proteins with which CtsX interacts. A CtsX-FLAG tagged protein has been made and is being used in these experiments. I will obtain a whole cell lysate under non-denaturing conditions to maintain protein-protein interactions. Using antibodies to the FLAG epitope tag, I will purify CtsX-FLAG and, presumably, any other protein associated with it. I will run the resulting protein complex on an acrylamide gel and stain to observe potential bands in the elution fractions. Finally, I will identify proteins associating with CtsX by MALDI-TOF analysis.