Cameron Woodward and William McCleary, Department of Microbiology and Molecular Biology

Introduction

Phosphate intake by Escherichia coli serves as a good model system to study phosphate intake by other bacteria. The pho phosphate uptake system used by E. coli is well conserved in pathogens like Vibrio cholerae (1). To better understand this important mode of phosphate uptake a study was performed to examine the interactions between the phoR and phoU proteins of this system. PhoU interacts with the pstSCAB complex, which brings phosphate into the cell. After communicating with pstSCAB the phoU protein interacts with phoR to tell it if phosphate is present in the environment or not. PhoR has been shown to be a membrane bound histidine kinase that phosphorylates and dephosphorylates the phoB protein to control expression of genes involved in phosphate uptake (2). The goal of this study was to insert mutations of the phoR gene into the E. coli chromosome in order to examine which amino acids are essential for interaction between phoU and phoR.

Methodology

The phoR gene was amplified using PCR using the plasmid pRR48phoR as a template. The cat chloramphenicol resistance gene was amplified by PCR from the plasmid pKD3. A region homologous to phoR (called HR from here on out) was added onto the end of the cat gene using a long PCR primer that contained the HR region as well as nucleotides with homology to cat. Both PCR products underwent PCR clean up using the kit from invitrogen. The cat and phoR gene products were then combined into one PCR product by using a forward PCR primer with homology to phoR and a reverse primer with homology to HR and cat. This complete phoR-cat-HR product was cleaned up using the invitrogen kit. This process was repeated many times using different template plasmids for the phoR gene. Each of these different template plasmids had site specific mutations on phoR, thus each final PCR product had a slightly different DNA sequence for the phoR gene. The presence of the complete PCR product was verified using restriction digests.

A strain of E. coli lacking the phoR gene, except a short sequence from the beginning and end of phoR, was obtained from another member of the McCleary lab. The complete PCR product was transformed into the ΔphoR strain through electroporation. Bacteria which received the target DNA sequence into the chromosome through recombination with the plasmid were selected for through growth on media containing chloramphenicol. To assure that these bacteria were ΔphoR, the bacteria were grown on low phosphate media containing the indicator xphos. On this media, it was expected that bacteria that cannot take in phosphate would grow up white while the wild type would grow up blue. The phoR-phoU interaction strength was measured qualitatively based on the color of colonies grown.

Results

The ΔphoR DNA sequence was created successfully, as well as the cat gene sequence. The two PCR products were combined successfully. However the ΔphoR strain of Escherichia coli, which was to be made by another member of the lab, could not be created for unknown reasons. The templates for the remaining mutated phoR sequences were to be made by another member of the lab. However, this member of the lab was not able to complete this part of the project due to his leaving the lab for personal reasons. Because of these obstacles, the site-specific mutations in the E.coli chromosome were not able to be introduced.

Discussion

Because the initial completed ΔphoR-cat PCR product could not be transformed into bacteria and the subsequent mutant phoR-cat PCR products could not be made, the amino acids essential to phoRphoU interaction could not be determined.

Conclusion

Sadly, the details of the phoR-phoU interaction could not elucidated. This experience shows the importance of the work of every member in a lab. While the procedure detailed here should be feasible to be carried out, because of our circumstances, they could not be performed by us. In the future, the McCleary lab hopes to take up a similar project again to better understand phoR-phoU interactions.

1. Lery, Letícia, Carolina L. Goulart, Felipe R. Figueiredo, Karine S. Verdoorn, Marcelo EinickerLamas, Fabio M. Gomes, Ednildo A. Machado, Paulo M. Bisch, and Wanda von Kruger. “A comparative proteomic analysis of Vibrio cholerae O1 wildtype cells versus a phoB mutant showed that the PhoB/PhoR system is required for full growth and rpoS expression under inorganic phosphate abundance.” Journal of proteomics 86 (2013): 115.

2. Gardner, Stewart G., Kristine D. Johns, Rebecca Tanner, and William R. McCleary. “The PhoU Protein from Escherichia coli Interacts with PhoR, PstB, and Metals To Form a Phosphate-Signaling Complex at the Membrane.” Journal of bacteriology 196, no. 9 (2014): 17411752.