| Project Description: Biofilms are three-dimensional structures made by bacteria that are characterized by pillars of bacteria interspersed with water channels. Biofilm formation is thought to play an important role in the survival of many bacteria including the aquatic pathogen Vibrio cholerae, which is the causative agent of the diarrheal disease cholera. Biofilm formation is an important feature of the survival of V. cholerae within the human host as well as its natural aquatic environments. Formation of biofilms are regulated by many different chemical signals in the environment. One of these signals is a group of small molecules called polyamines. The amount of various polyamines present within the cell as well as the external environment of the cell influences the formation of these biofilms.
Polyamines are important for cell survival and are essential to biofilm formation. In V. cholerae, multiple pathways involving transport and signaling networks affect the formation of biofilms. It has recently been found that the polyamine spermidine regulates biofilm through two different pathways. Spermidine can decrease biofilm formation through a signaling complex made up of the proteins NspS and MbaA without being transported into the cell. In addition spermidine is thought to be transported into the cell by the PotD1 protein. In this case a decrease in biofilms is also observed and this may be due to an intracellular interaction.
We have previously shown that when V. cholerae is grown in Luria-Bertani culture medium, which contains large amounts of spermidine, there are significant amounts of spermidine inside the cell. Because V. cholerae does not synthesize its own spermidine, all of the spermidine in the cell is transported from the external environment. We have also shown that the deletion of the PotD1 protein results in a complete loss of spermidine inside the cell. These results have suggested that PotD1 protein is correlated with the transport of spermidine into the cell. However, they are not sufficient to determine whether the deletion of the PotD1 protein directly diminishes the uptake of the polyamine spermidine or if there is a secondary indirect effect responsible for this observation. In order to confirm that PotD1 is directly responsible for spermidine transport a complementation experiment must be performed.
The purpose of the proposed study is to perform this complementation experiment to confirm the role of PotD1 in spermidine import. To do this, first a plasmid containing the potD1 gene will be first constructed. Then this plasmid will be inserted into back into the V. cholerae potD1 mutant which is missing the chromosomal copy of the potD1 gene. . Finally, this new strain will be grown in Luria-Bertani medium and the cellular polyamines will be quantified using high performance liquid chromatography. If the complemented strain regains the ability to import spermidine into the cell, this will suggest that PotD1 is directly responsible for spermidine transport. This study will further elucidate the functions of the PotD1 protein. |
