Michael Schurdell and Dr. William McCleary, Department of Microbiology and Molecular Biology
While researching the required amino acid residues of PhoU, we discovered a small intergenic region located between pstA and pstB on the pstSCAB-phoU operon. Defining the role of this small intergenic region took precedence over my previously proposed research project. All of my research on this project was performed in conjunction with Garrett Woodbury and William McCleary. We presented our findings in a poster presentation at the Intermountain Branch of the American Society of Microbiology Annual Meeting on Saturday, March 18, 2006. A peer reviewed article containing the full details of our work was accepted for publication on December 1, 2006 in the Journal of Bacteriology under the title, “Genetic evidence suggests that the intergenic region between pstA and pstB plays a role in the regulation of rpoS translation during phosphate limitation”.
Escherichia coli is constantly adapting to changing phosphate concentrations in its environment. The two component regulatory system, PhoB/PhoR, monitors phosphate availability by evaluating the activity of the Pst system . The Pst system is composed of a periplasmic binding protein, PstS, two cytoplasmic membrane bound proteins, PstA and PstC and an ATPase PstB found in the cytoplasm. A small intergenic region of interest exists between pstA and pstB. During phosphate starvation, the Pho system upregulates the expression of the Pho regulon through the autophosphorylation of the histidine kinase PhoR. PhoR then phosphorylates PhoB, which activates the Pho regulon. Mutations in the Pst system constitutively activate the Pho regulon via the same mechanism .
Under starvation conditions, E. coli enters stationary phase. The sigma factor responsible, in part, for the stress response that occurs under starvation conditions is RpoS. The accumulation of RpoS in the cell leads to the transcription of hundreds of stationary phase genes, including the gene that codes for hydrogen peroxidase II. The products of these genes make the cell resistant to a variety of environmental stresses including phosphate starvation. RpoS production is regulated at transcription, translation, protein stability, and activity1. Ruiz and Silhavy found that in pst mutants that constitutively activate the Pho regulon, the levels of RpoS are greatly elevated2. They propose that since the upregulation of rpoS translation was Hfq dependent a small regulatory RNA must be involved. The small intergenic region found between pstA and pstB shows some complementarity to the rpoS mRNA. We hypothesize that the above mentioned intergenic region binds to the rpoS mRNA and facilitates the translation of rpoS.
Bacterial strains and plasmids: Using the pKD3 plasmid, two mutant strains of E. coli were created: BM121 and BM122. Both mutants lacked the pstB and phoU genes to induce constitutive transcription of the Pho regulon. However, BM121 retained the small intergenic region downstream from pstA that is absent in BM122. The strain BW25113 served as a wild type control in all of our experiments and had no mutations to the Pho regulon. Both catalase and Western blot assays were used to determine relative amounts of RpoS in mutant and wild type samples. Catalase assay: BM121, BM122, and BW25113 strains were grown under the same conditions described above. During exponential growth phase, two samples were taken from each strain of cells. One set of samples was put on ice to stop continued growth and the second set was boiled to degrade the heat labile non-RpoS induced HPI. Samples that were not heated served as a control to measure total HP activity. Subjecting the heat shocked samples to a catalase assay allowed us to infer that the remaining HP was a result of the heat-stable RpoS induced HPII. The HPII activity served as an indicator of relative RpoS concentrations in the various strains of cells. Western blot: Strains BM121, BM122, and BW25113 were grown under aeration in 5 mL of LB broth at 37 degrees Celsius. When the cells reached an OD600 of 0.4 (representative of logarithmic growth phase) samples were then pelleted and resuspended in loading buffer. The resuspension volume in mL was equal to OD600/6. After boiling the resuspended cells for 10 minutes, the samples were loaded into a 12% SDS-polyacrylamide gel. The gel was subjected to electrophoresis and the proteins were then transferred onto a nitrocellulose membrane for Western blot analysis. Monoclonal mouse anti-RpoS antibody was used as a primary antibody. Wild type stationary phase cells were used as a control. The Western blot allowed us to compare relative amounts of RpoS produced in cells lacking the pstA-pstB intergenic region with cells that possessed the intergenic region.
To determine relative amounts of RpoS accumulation in mutant and wild type cells, we performed catalase and Western blot assays. The catalase assay performed during exponential phase shows that the BM121 mutant, which contains the small intergenic region in question, has higher levels of hydrogen peroxidase II activity than the BM122 mutant. Increased levels of HPII activity indirectly demonstrate an increase in cellular RpoS accumulation. To directly measure the accumulation of RpoS in the cell, we performed a Western blot. Results from the Western blot also indicate an increased level of RpoS in cells from the strain BM121 as compared to cells from the strain BM122.
Under starvation conditions, the cellular concentration of RpoS is increased in E. coli. The increase in RpoS leads to the transcription of hundreds of genes that prepare the cell for stationary phase. RpoS production is regulated on many levels, including transcription, translation, protein stability, activation1. Our results indicate that the small intergenic region located between pstA and pstB plays an important role in increasing the cellular concentration of RpoS by facilitating translation via complementary binding. By understanding the mechanisms involved in regulating the initiation of stationary phase, we can better understand how cells prepare for survival under stressful conditions.