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<OL> <LI> Define the process of Listeria monocytogenes biofilm development. It is clear that L. monocytogenes can form a single species biofilm on various surfaces that consist of large numbers of sessile bacteria. Our goal is to define the three-dimensional dynamics of biofilm formation using a flow-cell apparatus in combination with phase-contrast and confocal laser scanning microscopy. This analysis will reveal the pathway that leads to biofilm formation and will reveal the stages of biofilm development. This information will be used to provide the foundation for evaluating the specific role of Listeria genes identified in Specific Objective 2 to be essential for biofilm development. <LI> Define structural elements and genes that are required for L. monocytogenes biofilm development. It is important to identify structural components, regulatory pathways and physiological activities that contribute to biofilm formation. We have already established a genetic approach that will provide valuable information toward our goal of identifying genes essential for biofilm development (ebd). These genes will be characterized to define their functions and to evaluate their role in the process of biofilm formation. <LI> Utilize DNA microarray analysis to define the genes expressed during the biofilm maturation process on an abiotic surface. This effort will complement specific objective 1 and 2 by defining the specific stage at which genes contributing to biofilm development are expressed. It will also allow for the estimation of how gene regulatory circuits combine to control physical and physiological activities of Listeria during biofilm maturation.
APPROACH: Specific Objective 1. Define the process of Listeria monocytogenes biofilm development. To reveal the dynamic nature of Listeria biofilm formation, we propose to examine Listeria biofilm development using two microscopy techniques in combination with growth of Listeria biofilms using a flow cell apparatus. Biofilm development can be directly and non-destructively monitored by microscopy. First we will use light and phase contrast microscopy to evaluate the initial stages of biofilm formation and to generate a low-resolution image of biofilm development and maturation. The results of phase-contrast microscopy will provide a foundation for our second approach which will be to use confocal laser scanning microscopy to define the three dimensional structure of Listeria biofilms at each of the stages of development. Specific Objective 2. Define structural elements and genes that are required for L. monocytogenes biofilm development. We will use a simple genetic screen that utilizes the well of a microtiter dish as the chamber in which a biofilm is established. Biofilms are visualized by staining with crystal violet dye. The simplicity of this assay means that high-throughput screens of many thousands of randomly generated mutants can be carried out with relative ease. A 9600 member transposon library using the transposon Tn917-LTV1 will be screened. By identifying biofilm developmental genes, we will gain insight as to the type of surface structures, physiological pathways and regulatory mechanisms that are important for Listeria to adapt from a planktonic state to a lifestyle as an attached microbial community. Specific Objective 3. Utilize DNA microarray analysis to define the genes expressed during the biofilm maturation process on an abiotic surface. A time course will be performed extending over 48 hours to survey gene expression at times reflecting different stages of biofilm development. Total RNA from Listeria biofilms will be isolated from six replicates for each of the five time points (0, 4, 12, 24 and 48 h). RNA will be converted to cDNA and labeled using established methods. Two labeled products will be mixed and added to the microarrays. Data will be analyzed by established methods and total gene expression over the time course compared. Documents SCA with UC Davis.
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PROGRESS: 2006/07 TO 2009/06 <BR>
The period of performance for this project with the Principle Investigator at UC Davis was extended for one more year to end on June 30, 2009. The project is progressing with a paper submitted on the analysis of a mutant library of L. monocytogenes for biofilm formation mutants. More work is needed to determine a genetic basis for biofilm development. ADODR monitoring was done by frequent e-mails and visits between the UC Davis and ARS Labs. This research addresses National Program 108 problem statement 1.2.3. (Production and Processing Ecology).