Isoprenoids are essential in all living organisms. Cells require them for respiration, membrane biosynthesis, and proliferation. In Escherichia coli, isoprenoids are used to synthesize the lipid carrier undecaprenyl phosphate (Und-P), which transports peptidoglycan (PG) intermediates for cell wall biosynthesis. Since Und-P is essential for PG biosynthesis, decreasing the available Und-P for transporting intermediates can cause shape and growth defects. Although Und-P synthesis is well understood, it is unknown how much Und-P is needed to maintain normal cell shape and growth, or how decreased Und-P levels may affect other cellular functions. In this work, I characterize a missense mutation in undecaprenyl phosphate synthase (uppS) (Chapter 2), describe phenotypes associated with low levels of undecaprenyl phosphate (Und-P) (Chapter 3), and focus on developing protocols to measure Und-P levels in vivo (Chapter 4).
A missense mutation in undecaprenyl pyrophosphate synthase results in a defective UppS, which contributes to cells bulging and filamenting and to growth defects. These defects were suppressed by compensatory mutations upstream of uppS in the isoprenoid biosynthetic pathway. These findings suggest that cells with a defective UppS will accumulate mutations that increase isoprenoid production for PG biosynthesis. Also, increasing the substrates available to the defective UppS suppressed the shape defects caused by low levels of Und-P. In short, cells are highly sensitive to changes in isoprenoid levels.
We used the knowledge gained from the preceding experiments to identify growth defects associated with low levels of Und-P. In E. coli, PG synthesis depends on Und-P for transporting PG precursors. In the absence of the major PG synthase, PBP1B, a subpopulation of cells with a defective UppS lyse at elevated temperature. Alternatively, chemically inhibiting Und-P production lyses cells that lack PBP1B. Cells suppressed these growth defects by increasing the flux of Und-P to PG biosynthesis at the expense of another Und-P utilizing pathway or by increasing Und-P levels. These findings suggest that disrupting PG biosynthesis in combination with low levels of Und-P can prime cells for lysis.
Finally, we quantified isoprenoid and Und-P levels in E. coli. Since there is no easy assay available to quantify in vivo Und-P levels, we designed protocols to indirectly and directly measure these levels. We determined that one strain of E. coli, MG1655, has more available isoprenoids than another strain, CS109. Also, cells carrying a Ser-to-Ala replacement at aa 269 in IspH were more resistant to an isoprenoid inhibitor. Using these assays, we furthered our understanding of how alterations in Und-P synthesis affect isoprenoid availability.
Taken together, this work indicates that cells are highly susceptible to shape and growth defects when Und-P is limited. Also, we suggest that there is a hierarchy for partitioning Und-P among competing Und-P dependent pathways.
|Advisor:||Young, Kevin D.|
|Commitee:||Blevins, Jon S., Boehme, Karl W., Kelly, Thomas J., Voth, Daniel E.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Microbiology and Immunology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 81/2(E), Dissertation Abstracts International|
|Keywords:||Escherichia coli, Isoprenoids, Metabolism, Morphology, Und-P, UppS|
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