Svetlana Alexeeva

The integral membrane FtsW protein and peptidoglycan synthase PBP3 form a subcomplex in Escherichia coli

During the cell cycle of rod-shaped bacteria, two morphogenetic processes can be discriminated: length growth of the cylindrical part of the cell and cell division by formation of two new cell poles. The morphogenetic protein complex responsible for the septation during cell division (the divisome) includes class A and class B penicillin-binding proteins (PBPs). In Escherichia coli, the class B PBP3 is specific for septal peptidoglycan synthesis. It requires the putative lipid II flippase FtsW for its localization at the division site and is necessary for the midcell localization of the class A PBP1B. In this work we show direct interactions between FtsW and PBP3 in vivo and in vitro by FRET (Förster resonance energy transfer) and co-immunoprecipitation experiments. These proteins are able to form a discrete complex independently of the other cell-division proteins. The K2-V42 peptide of PBP3 containing the membrane-spanning sequence is a structural determinant sufficient for interaction with FtsW and for PBP3 dimerization. By using a two-hybrid assay, the class A PBP1B was shown to interact with FtsW. However, it could not be detected in the immunoprecipitated FtsW-PBP3 complex. The periplasmic loop 9/10 of FtsW appeared to be involved in the interaction with both PBP1B and PBP3. It might play an important role in the positioning of these proteins within the divisome.

DNA and origin region segregation are not affected by the transition from rod to sphere after inhibition of Escherichia coli MreB by A22.

The bacterial actin homologue MreB forms a helix underneath the cytoplasmic membrane and was shown to be essential in the morphogenesis of the rod‐shaped cells. Additionally, MreB was implicated to be involved in DNA segregation. However, in our hands the mreBCD deletion strain (PA340‐678) grew without apparent DNA segregation defect, suggesting that the reported chromosome segregation inhibition could be caused by a temporarily effect of MreB inhibition or depletion. To assess the involvement of MreB in DNA segregation during the transition from rod to sphere, we compared the effect of A22 and the PBP2 inhibitor mecillinam on the percentage of cells with segregated nucleoids and the number of oriC foci in wild‐type Escherichia coli cells. Cells became spherical in the same time window during both treatments and we could not detect any difference in the chromosome or oriC segregation between these two treatments. Additionally, flow cytometric analyses showed that A22 and mecillinam treatment gave essentially the same chromosome segregation pattern. We conclude that MreB is not directly involved in DNA segregation of E. coli.

Colocalization and interaction between elongasome and divisome during a preparative cell division phase in Escherichia coli.

The rod‐shaped bacterium Escherichia coli grows by insertion of peptidoglycan into the lateral wall during cell elongation and synthesis of new poles during cell division. The monofunctional transpeptidases PBP2 and PBP3 are part of specialized protein complexes called elongasome and divisome, respectively, which catalyse peptidoglycan extension and maturation. Endogenous immunolabelled PBP2 localized in the cylindrical part of the cell as well as transiently at midcell. Using the novel image analysis tool Coli‐Inspector to analyse protein localization as function of the bacterial cell age, we compared PBP2 localization with that of other E. coli cell elongation and division proteins including PBP3. Interestingly, the midcell localization of the two transpeptidases overlaps in time during the early period of divisome maturation. Försters Resonance Energy Transfer (FRET) experiments revealed an interaction between PBP2 and PBP3 when both are present at midcell. A decrease in the midcell diameter is visible after 40% of the division cycle indicating that the onset of new cell pole synthesis starts much earlier than previously identified by visual inspection. The data support a new model of the division cycle in which the elongasome and divisome interact to prepare for cell division.

Use of thymine limitation and thymine starvation to study bacterial physiology and cytology.

A variety of very basic questions asked 30 to 40 years ago remain unanswered today. How do bacteria select and maintain a defined size, length, and width? How do they control the timing of chromosome replication? What is the mechanism by which they segregate their replicated (or replicating)

Differential Bacterial Surface Display of Peptides by the Transmembrane Domain of OmpA

Peptide libraries or antigenic determinants can be displayed on the surface of bacteria through insertion in a suitable outer membrane scaffold protein. Here, we inserted the well-known antibody epitopes 3xFLAG and 2xmyc in exterior loops of the transmembrane (TM) domain of OmpA. Although these highly charged epitopes were successfully displayed on the cell surface, their levels were 10-fold reduced due to degradation. We verified that the degradation was not caused by the absence of the C-terminal domain of OmpA. In contrast, a peptide that was only moderately charged (SA-1) appeared to be stably incorporated in the outer membrane at normal protein levels. Together, these results suggest that the display efficiency is sensitive to the charge of the inserted epitopes. In addition, the high-level expression of OmpA variants with surface-displayed epitopes adversely affected growth in a strain dependent, transient manner. In a MC4100 derived strain growth was affected, whereas in MC1061 derived strains growth was unaffected. Finally, results obtained using a gel-shift assay to monitor β-barrel folding in vivo show that the insertion of small epitopes can change the heat modifiability of the OmpA TM domain from ‘aberrant’ to normal, and predict that some β-barrels will not display any significant heat-modifiability at all.