H-050. Identification of Interactions within the GacA/S Two-Component Regulatory System of Pseudomonas fluorescens CHA0

L. Chang, M. L. Workentine, H. Ceri, R. J. Turner;
Univ. of Calgary, Calgary, AB, CANADA.

The two-component regulatory system comprised of the sensor kinase, GacS, and its response regulator, GacA, is involved in regulation of secondary metabolism and many aspects of bacterial physiology such as biofilm formation, virulence, quorum sensing, and phenotypic variation. This system regulates the production of small regulatory RNAs which relieves the repression of RsmA/E on target genes. It is also known that other sensor kinases such as RetS and LadS may feed into this system. To further characterize the protein-protein interactions in the GacA/S system we utilized a bacterial two-hybrid (BACTH) system to demonstrate interactions between the two-component regulators GacS and GacA of Pseudomonas fluorescens. Domains of GacA and GacS, identified through bioinformatics, were subcloned and their ability to interact in-vivo was investigated. Results of this study indicate that the entire GacA molecule is required for GacA interacting with itself or GacS. Furthermore, the HisKA/HATPase/REC domains of GacS together are responsible for GacS interacting with GacA, while the HAMP domain of GacS is responsible for GacS interacting with itself. The BACTH system was also used to screen a library from P. fluorescens for additional proteins interacting with GacA and GacS. Although no GacA interacting proteins were identified in this study other than GacS, we have identified two uncharacterized proteins that interact with GacS. These proteins are: PFL_2537, putatively identified as a methyl-accepting chemo taxis protein (MCP), and PFL_5051, a potential heavy metal sensor histidine kinase (HemS). In this study we have presented an in-vivo characterization of the interactions of GacS and GacA and we have identified novel interactors with GacS, giving further evidence that this system may contain far more than two components.