Biology subjects

den Blaauwen, T.

Publications and source records attributed to den Blaauwen, T..

4 recordsLinked to original sources

bioRxiv2018

The bacterial DNA binding protein MatP involved in linking the nucleoid terminal domain to the divisome at midcell interacts with lipid membranes

Division ring formation at midcell is controlled by various mechanisms in Escherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipids in vitro. We found that MatP, when encapsulated inside microdroplets generated by microfluidics and giant vesicles, accumulates at phospholipid bilayers and monolayers matching the lipid composition in the E. coli inner membrane. MatP binding to lipids was independently confirmed using lipid coated microbeads and bio-layer interferometry assays. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the interaction of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands.\n\nIMPORTANCEThe division of an E. coli cell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integrated in vivo and in vitro analysis provides evidence that MatP can interact with lipid membranes comprising the phospholipid mixture in the E. coli inner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division.

biochemistry↗

bioRxiv2018

Superfolder mTurquoise2ox optimized for the bacterial periplasm allows high efficiency in vivo FRET of cell division antibiotic targets.

Fluorescent proteins (FP)s are of vital importance to biomedical research. Many of the currently available fluorescent proteins do not fluoresce when expressed in non-native environments, such as the bacterial periplasm. This strongly limits the options for applications that employ multiple FPs, such as multiplex imaging or FRET. To address this issue, we have engineered a new cyan fluorescent protein based on mTurquoise2 (mTq2). The new variant is dubbed superfolder turquoise 2 ox (sfTq2ox) and is able to withstand challenging, oxidizing environments. sfTq2ox has improved folding capabilities and can be expressed in the periplasm at higher concentrations without toxicity. This was tied to the replacement of native cysteines that may otherwise form promiscuous disulfide-bonds. The improved sfTq2ox has the same spectroscopic properties as mTq2, i.e. high fluorescence lifetime and quantum yield. The sfTq2ox-mNeongreen FRET pair allows the detection of periplasmic protein-protein interactions with energy transfer rates exceeding 40 %. Employing the new FRET pair, we show the direct interaction of two essential periplasmic cell division proteins FtsL and FtsB and disrupt it by mutations, paving the way for in vivo antibiotic screening.

microbiology↗

bioRxiv2018

Structural analysis of the interaction between the bacterial cell division proteins FtsQ and FtsB

Most bacteria and archaea use similar proteins within their cell division machinery, which uses the tubulin homologue FtsZ as its central organiser. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. In biochemical assays we show that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the {beta} domain of FtsQ shows that only residues 64-87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ:FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ:FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalising view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches.\n\nImportanceCells in most bacteria and archaea divide through a cell division process that is characterised through its filamentous organiser, FtsZ protein. FtsZ forms a ring structure at the division site and starts the recruitment of 10-20 downstream proteins that together form an elusive multi-protein complex termed divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.

microbiology↗

bioRxiv2017

The Combined activities of FtsW and MurJ are required for lipid II translocation during cell division in Escherichia coli

Peptidoglycan (PG) is the unique cell shape-determining component of the bacterial envelope, and is a key target for antibiotics. PG synthesis requires the transmembrane movement of the precursor lipid II, and MurJ has been shown to provide this activity in E. coli. However, how MurJ functions in vivo has not been reported. Here we show that MurJ localizes both in the lateral membrane and at midcell, and is recruited to midcell simultaneously with late-localizing divisome proteins and proteins MraY and MurG. MurJ septal localization is dependent on the presence of a complete and active divisome, lipid II synthesis and PBP3/FtsW activities. Inactivation of MurJ, either directly by mutation or through binding with MTSES, did not affect the midcell localization of MurJ. Our study visualizes MurJ localization in vivo and reveals a possible mechanism of how MurJ functions during cell division, which gives possibilities for future investigations and further antibiotics developments.

microbiology↗