This application is to explore the function in attachment and biofilm formation of cluster II chemotaxis-like genes found in the opportunistic human pathogen Pseudomonas aeruginosa. P. aeruginosa has multiple sets of chemotaxis-like genes arranged in clusters. Genes in clusters I/V are absolutely required for flagella-mediated chemotaxis. Those in cluster IV are required for pilus-mediated motility and response. Accumulating evidence suggests that some chemotaxis-like genes from bacteria may not be primarily involved in chemotaxis or motility. Such appears to be the case for P. aeruginosa cluster II genes. Cluster II che-like and mcp-like genes are involved in attachment of cells to surfaces and biofilm formation. They appear to play, at most, a minor role in flagella-mediated chemotaxis. Cluster II mutants are not defective in pilus-mediated twitching motility or in swarming across solid surfaces. The long-term objective of the proposed work is to determine the mechanism by which a presumed cluster II signal transduction complex directs cells to attach to surfaces and initiate biofilm formation.
In specific aim 1 P. aeruginosa gene chips will be used to identify sets of genes that are transcriptionally activated or repressed by cluster II che-like proteins. Other experiments will explore the possibility that a cluster II signaling complex has a major role in directly modulating the activity of a cell surface molecular machine during the stationary phase of growth. The possibility that cluster II proteins modulate the activities of other proteins through direct physical contact will also be examined.
In specific aim 2 genes identified as being regulated by cluster II signal transduction or genes encoding proteins whose activities are modulated by cluster II proteins, will be inactivated by mutation. The attachment and biofilm formation phenotypes of the mutants will be analyzed. Complementary physiological approaches will be used to explore how the activities of the encoded proteins may affect biofilm formation. Cluster II genes are induced in the stationary phase of growth in a cell-density dependant manner.
Specific aim 3 will explore the hypothesis that cluster II genes and somemcp genes are coordinately regulated by global regulators that control the exponential-to-stationary phase growth transition in P. aeruginosa. The hypothesis that cluster II proteins interact to form a signal transduction complex will be tested in specific aim 4. Phospho-transfer assays will be carried out and effects of cluster II proteins on the subcellular localization of other green fluorescent tagged cluster II proteins will be determined. Pseudomonas aeruginosa grows as a biofilm in the lungs of cystic fibrosis patients and it grows as a biofilm in infections caused by indwelling medical devices. The proposed work will reveal proteins and genes that are required for biofilm development by prokaryotes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056665-08
Application #
6944034
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Rodewald, Richard D
Project Start
1998-05-01
Project End
2007-08-31
Budget Start
2005-09-01
Budget End
2006-08-31
Support Year
8
Fiscal Year
2005
Total Cost
$299,313
Indirect Cost
Name
University of Washington
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Baraquet, Claudine; Harwood, Caroline S (2016) FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa. J Bacteriol 198:178-86
Martín-Moldes, Zaira; Blázquez, Blas; Baraquet, Claudine et al. (2016) Degradation of cyclic diguanosine monophosphate by a hybrid two-component protein protects Azoarcus sp. strain CIB from toluene toxicity. Proc Natl Acad Sci U S A 113:13174-13179
Matsuyama, Bruno Y; Krasteva, Petya V; Baraquet, Claudine et al. (2016) Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 113:E209-18
Chen, Annie I; Dolben, Emily F; Okegbe, Chinweike et al. (2014) Candida albicans ethanol stimulates Pseudomonas aeruginosa WspR-controlled biofilm formation as part of a cyclic relationship involving phenazines. PLoS Pathog 10:e1004480
Baraquet, Claudine; Harwood, Caroline S (2013) Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ. Proc Natl Acad Sci U S A 110:18478-83
Huangyutitham, Varisa; Guvener, Zehra Tuzun; Harwood, Caroline S (2013) Subcellular clustering of the phosphorylated WspR response regulator protein stimulates its diguanylate cyclase activity. MBio 4:e00242-13
Baraquet, Claudine; Murakami, Keiji; Parsek, Matthew R et al. (2012) The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP. Nucleic Acids Res 40:7207-18
Chugani, Sudha; Kim, Byoung Sik; Phattarasukol, Somsak et al. (2012) Strain-dependent diversity in the Pseudomonas aeruginosa quorum-sensing regulon. Proc Natl Acad Sci U S A 109:E2823-31
Irie, Yasuhiko; Borlee, Bradley R; O'Connor, Jennifer R et al. (2012) Self-produced exopolysaccharide is a signal that stimulates biofilm formation in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 109:20632-6
O'Connor, Jennifer R; Kuwada, Nathan J; Huangyutitham, Varisa et al. (2012) Surface sensing and lateral subcellular localization of WspA, the receptor in a chemosensory-like system leading to c-di-GMP production. Mol Microbiol 86:720-9

Showing the most recent 10 out of 26 publications