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Mutants, suppressors, and wrinkled colonies: mutant alleles of the cell division gene ftsQ point to functional domains in FtsQ and a role for domain 1C of FtsA in divisome assembly.

ABSTRACT: Cell division in Escherichia coli requires the concerted action of at least 10 essential proteins. One of these proteins, FtsQ, is physically associated with multiple essential division proteins, including FtsK, FtsL, FtsB, FtsW, and FtsI. In this work we performed a genetic analysis of the ftsQ gene. Our studies identified C-terminal residues essential for FtsQ's interaction with two downstream proteins, FtsL and FtsB. Here we also describe a novel screen for cell division mutants based on a wrinkled-colony morphology, which yielded several new point mutations in ftsQ. Two of these mutations affect localization of FtsQ to midcell and together define a targeting role for FtsQ's alpha domain. Further characterization of one localization-defective mutant protein [FtsQ(V92D)] revealed an unexpected role in localization for the first 49 amino acids of FtsQ. Finally, we found a suppressor of FtsQ(V92D) that was due to a point mutation in domain 1C of FtsA, a domain previously implicated in the recruitment of divisome proteins. However, despite reports of a potential interaction between FtsA and FtsQ, suppression by FtsA(I143L) is not mediated via direct contact with FtsQ. Rather, this mutation acts as a general suppressor of division defects, which include deletions of the normally essential genes zipA and ftsK and mutations in FtsQ that affect both localization and recruitment. Together, these results reveal increasingly complex connections within the bacterial divisome.

SUBMITTER: Goehring NW

PROVIDER: S-EPMC1797389 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Mutants, suppressors, and wrinkled colonies: mutant alleles of the cell division gene ftsQ point to functional domains in FtsQ and a role for domain 1C of FtsA in divisome assembly.

Goehring Nathan W NW Petrovska Ivana I Boyd Dana D Beckwith Jon J

Journal of bacteriology 20060915 2

Cell division in Escherichia coli requires the concerted action of at least 10 essential proteins. One of these proteins, FtsQ, is physically associated with multiple essential division proteins, including FtsK, FtsL, FtsB, FtsW, and FtsI. In this work we performed a genetic analysis of the ftsQ gene. Our studies identified C-terminal residues essential for FtsQ's interaction with two downstream proteins, FtsL and FtsB. Here we also describe a novel screen for cell division mutants based on a wr ...[more]

PMID: 16980443

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Project description:BACKGROUND: Bacterial division is produced by the formation of a macromolecular complex in the middle of the cell, called the divisome, formed by more than 10 proteins. This process can be divided into two steps, in which the first is the polymerization of FtsZ to form the Z ring in the cytoplasm, and then the sequential addition of FtsA/ZipA to anchor the ring at the cytoplasmic membrane, a stage completed by FtsEX and FtsK. In the second step, the formation of the peptidoglycan synthesis machinery in the periplasm takes place, followed by cell division. The proteins involved in connecting both steps in cell division are FtsQ, FtsB and FtsL, and their interaction is a crucial and conserved event in the division of different bacteria. These components are small bitopic membrane proteins, and their specific function seems to be mainly structural. The purpose of this study was to obtain a structural model of the periplasmic part of the FtsB/FtsL/FtsQ complex, using bioinformatics tools and experimental data reported in the literature. RESULTS: Two oligomeric models for the periplasmic region of the FtsB/FtsL/FtsQ E. coli complex were obtained from bioinformatics analysis. The FtsB/FtsL subcomplex was modelled as a coiled-coil based on sequence information and several stoichiometric possibilities. The crystallographic structure of FtsQ was added to this complex, through protein-protein docking. Two final structurally-stable models, one trimeric and one hexameric, were obtained. The nature of the protein-protein contacts was energetically favourable in both models and the overall structures were in agreement with the experimental evidence reported. CONCLUSIONS: The two models obtained for the FtsB/FtsL/FtsQ complex were stable and thus compatible with the in vivo periplasmic complex structure. Although the hexameric model 2:2:2 has features that indicate that this is the most plausible structure, the ternary complex 1:1:1 cannot be discarded. Both models could be further stabilized by the binding of the other proteins of the divisome. The bioinformatics modelling of this kind of protein complex, whose function is mainly structural, provide useful information. Experimental results should confirm or reject these models and provide new data for future bioinformatics studies to refine the models.

Project description:Most bacteria and archaea use the tubulin homologue FtsZ as its central organizer of cell division. 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. We show in biochemical assays 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 ? domain of FtsQ shows that only residues 64 to 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 tantalizing view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches.IMPORTANCE In most bacteria and archaea, filaments of FtsZ protein organize cell division. FtsZ forms a ring structure at the division site and starts the recruitment of 10 to 20 downstream proteins that together form a multiprotein complex termed the 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 in detail how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.

Dataset Information

Mutants, suppressors, and wrinkled colonies: mutant alleles of the cell division gene ftsQ point to functional domains in FtsQ and a role for domain 1C of FtsA in divisome assembly.

Publications

Mutants, suppressors, and wrinkled colonies: mutant alleles of the cell division gene ftsQ point to functional domains in FtsQ and a role for domain 1C of FtsA in divisome assembly.

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