Bacterial cell shape is defined by a stress-bearing exoskeletal cell wall consisting of peptidoglycan (PG). Polymerization and maturation of PG requires a group of proteins called penicillin binding proteins (PBPs), which perform PG synthesis and modification. In Escherichia coli, a set of PG modifying PBPs, the D,D-carboxypeptidases (D,DCPases) play a crucial role in maintaining the cell’s rod shape, and mutants lacking these proteins produce branches, buds and kinks. To discover the origin of these branches, we followed the growth and division of these mutants and found that branches arose from abnormal septation events, suggesting that D,D-CPases were involved in cytokinesis. Since PBP5 is the major D,D-CPase in E. coli, we tried to localize this protein by fusing it to the carboxy terminus of TorA-GFP, which allows fusion proteins to be exported through the Twin Arginine Translocation (TAT) pathway. However, the fusion was not exported to the periplasm; instead, the fusion protein clogged the TAT translocon. To circumvent this problem, PBP5 and other D,D-CPases were co-translationally exported to the periplasm by fusing each one to superfolder-GFP, a periplasmic folding variant of GFP. Fusions to PBP5, PBP6 and DacD localized to the septum in a substrate dependent manner. Surprisingly, FtsZ formed abnormal helical and tilted structures in mutants lacking the D,D-CPases. These abnormal Z-rings were responsible for branch formation because they directed new PG synthesis in an abnormal orientation. These observations suggest that the periplasmic D,D-CPases fine-tune cytokinesis by regulating the formation or geometric placement of the FtsZ division ring.
In E. coli, PBP3 is the only PG synthase known to be essential for cell division, but it only catalyzes a transpeptidation reaction. However, PG polymerization also requires a glycosyltransferase activity, suggesting that other PG synthesizing PBPs also participate in cell division. In support of this view, a ring of new PG is synthesized at the division site in an FtsZ-dependent manner, even before the recruitment of PBP3. Formation of this new PG is called PBP3-independent PG synthesis (PIPS). Since FtsZ is cytoplasmic and the PG synthases are periplasmic, we hypothesized that these proteins interact via an integral membrane protein(s). Known cell division proteins were screened to identify their participation in PIPS, and we found that ZipA, an FtsZ-interacting integral membrane protein, was required for PIPS. However, PIPS was still observed in a strain that bypasses the requirement of ZipA, arguing against the role of ZipA as an inner membrane connector. Using mutants that lack specific PBPs, we also checked the role of other PG synthases in PIPS. However, because of their redundant nature, it was not possible to determine the involvement of a specific PBP in this process. It remains to be determined how FtsZ directs PG synthesis via ZipA in wild type E. coli.
Overall, our results show that branches in E. coli arise from abnormal cell division, and periplasmic D,D-CPases play a cruicial role in fine-tuning the assembly and orientation of cytoplasmic FtsZ ring. In addition, we show that ZipA is required for pre-septal PG synthesis in wild type E. coli.
|Advisor:||Young, Kevin D.|
|Commitee:||Lee, Chiya Y., Morrison, Richard P., Varughese, Kottayil I., Voth, Daniel E.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Microbiology and Immunology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 73/05, Dissertation Abstracts International|
|Subjects:||Cellular biology, Microbiology, Biochemistry|
|Keywords:||Cell division, Cell shapes, Escherichia coli, Ftsz, Penicillin binding proteins, Peptidoglycan, Super folder gfp|
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