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Biomedical subjects

L I Rothfield

Publications and source records attributed to L I Rothfield.

At least 19 recordsLinked to original sources
PubMed2001

The MinE ring required for proper placement of the division site is a mobile structure that changes its cellular location during the Escherichia coli division cycle.

Placement of the division site at midcell in Escherichia coli requires the MinE protein. MinE acts by imparting topological specificity to the MinCD division inhibitor, preventing the inhibitor from acting at the midcell site while permitting it to block division at other unwanted sites along the length of the cell. It was previously shown that MinE assembled into a ring structure that appeared to be localized near midcell, apparently explaining the ability of MinE to specifically counteract MinCD at midcell. We report here that the MinE ring is not fixed in position near midcell but is a dynamic structure that undergoes a repetitive cycle of movement first to one cell pole and then to the opposite pole. Taken together with studies of the dynamic behavior of the MinD protein, the results suggest that the topological specificity of division site placement may not involve a localized action of MinE to counteract the MinCD division inhibitor at midcell but rather the ability of MinE to move the division inhibitor away from midcell and to the cell poles.

Adenosine Triphosphatases↗
PubMed2000

Structural basis for the topological specificity function of MinE.

Correct positioning of the division septum in Escherichia coli depends on the coordinated action of the MinC, MinD and MinE proteins. Topological specificity is conferred on the MinCD division inhibitor by MinE, which counters MinCD activity only in the vicinity of the preferred midcell division site. Here we report the structure of the homodimeric topological specificity domain of Escherichia coli MinE and show that it forms a novel alphabeta sandwich. Structure-directed mutagenesis of conserved surface residues has enabled us to identify a spatially restricted site on the surface of the protein that is critical for the topological specificity function of MinE.

Amino Acid Sequence↗
PubMed2000

Cell division inhibitors SulA and MinC/MinD block septum formation at different steps in the assembly of the Escherichia coli division machinery.

SulA and MinCD are specific inhibitors of cell division in Escherichia coli. In this paper, size exclusion chromatography was used to study the effect of the SulA and MinCD division inhibitors on the oligomerization state of endogenous FtsZ in cytoplasmic extracts, and immunofluorescence microscopy was used to determine the effect of SulA and MinCD on the formation of FtsZ, FtsA and ZipA rings at potential division sites. SulA prevented the formation of high-molecular-weight FtsZ polymers by interfering with FtsZ dimerization and subsequent oligomerization. In contrast, the MinCD division inhibitor did not prevent the oligomerization of FtsZ in the cell extracts or the formation of FtsZ and ZipA ring structures in vivo. However, MinCD did prevent the formation of FtsA rings. Increased expression of ftsA suppressed MinCD-induced division inhibition, but had no effect on SulA-induced division inhibition. These results indicate that MinCD blocks the assembly of the septation machinery at a later step than SulA, at the stage at which FtsA is added to the FtsZ ring.

Adenosine Triphosphatases↗
PubMed2000

Membrane redistribution of the Escherichia coli MinD protein induced by MinE.

Escherichia coli cells contain potential division sites at midcell and adjacent to the cell poles. Selection of the correct division site at midcell is controlled by three proteins: MinC, MinD, and MinE. It has previously been shown (D. Raskin and P. de Boer, Cell 91:685-694, 1997) that MinE-Gfp localizes to the midcell site in an MinD-dependent manner. We use here Gfp-MinD to show that MinD associates with the membrane around the entire periphery of the cell in the absence of the other Min proteins and that MinE is capable of altering the membrane distribution pattern of Gfp-MinD. Studies with the isolated N-terminal and C-terminal MinE domains indicated different roles for the two MinE domains in the redistribution of membrane-associated MinD.

Adenosine Triphosphatases↗
PubMed1999

The dimerization and topological specificity functions of MinE reside in a structurally autonomous C-terminal domain.

Correct placement of the division septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non-specific division inhibitor that blocks septation at all potential division sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles. Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88. Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE1-22 indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations. This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD.

Amino Acid Sequence↗
PubMed1999

Nucleoid-independent identification of cell division sites in Escherichia coli.

The mechanism used by Escherichia coli to determine the correct site for cell division is unknown. In this report, we have attempted to distinguish between a model in which septal position is determined by the position of the nucleoids and a model in which septal position is predetermined by a mechanism that does not involve nucleoid position. To do this, filaments with extended nucleoid-free regions adjacent to the cell poles were produced by simultaneous inactivation of cell division and DNA replication. The positions of septa that formed within the nucleoid-free zones after division was allowed to resume were then analyzed. The results showed that septa were formed at a uniform distance from cell poles when division was restored, with no relation to the distance from the nearest nucleoid. In some cells, septa were formed directly over nucleoids. These results are inconsistent with models that invoke nucleoid positioning as the mechanism for determining the site of division site formation.

Bacterial Proteins↗
PubMed1996

An extracellular factor regulates expression of sdiA, a transcriptional activator of cell division genes in Escherichia coli.

The sdiA gene codes for a protein that regulates expression of the ftsQAZ cluster of essential cell division genes of Escherichia coli. SdiA up-regulates the ftsQ2p promoter that initiates transcription into the ftsQAZ cluster. In this paper, we report that expression of sdiA is itself regulated by a factor that is released into the growth medium by E. coli. When medium that had previously supported growth of E. coli (conditioned medium) was used to support growth of an indicator E. coli strain that contained an sdiA-lacZ transcriptional reporter, there was a 50 to 80% decrease in sdiA expression as monitored by beta-galactosidase activity. The down-regulation of PsdiA was associated with a decrease in expression of the SdiA target promoter ftsQ2p, as monitored by expression of an ftsQ2p-lacZ transcriptional fusion. An effect of conditioned medium on ftsQ2p expression was not seen when the wild-type sdiA gene was disrupted by insertional mutagenesis, indicating that the effect on ftsQ2p expression was secondary to the down-regulation of PsdiA. Conditioned medium had no effect on expression of Plac, PrpoS, or several other promoters associated with the ftsQAZ gene cluster (ftsQ1p and ftsZ1-4p). This suggests that the response is specific for PsdiA and for promoters that are regulated by the sdiA gene product and that cell-to-cell signalling may play a role in regulating expression of this group of genes.

4-Butyrolactone↗
PubMed1995

Proper placement of the Escherichia coli division site requires two functions that are associated with different domains of the MinE protein.

The proper placement of the Escherichia coli division septum requires the MinE protein. MinE accomplishes this by imparting topological specificity to a division inhibitor coded by the minC and minD genes. As a result, the division inhibitor prevents septation at potential division sites that exist at the cell poles but permits septation at the normal division site at midcell. In this paper, we define two functions of MinE that are required for this effect and present evidence that different domains within the 88-amino acid MinE protein are responsible for each of these two functions. The first domain, responsible for the ability of MinE to counteract the activity of the MinCD division inhibitor, is located in a small region near the N terminus of the protein. The second domain, required for the topological specificity of MinE function, is located in the more distal region of the protein and affects the site specificity of placement of the division septum even when separated from the domain responsible for suppression of the activity of the division inhibitor.

Adenosine Triphosphatases↗
PubMed1994

Early stages in development of the Escherichia coli cell-division site.

Development of the Escherichia coli cell division site was studied in wild-type cells and in non-septate filaments of ftsZnull and ftsZTs mutant cells. Localized regions of plasmolysis were used as markers for the positions of annular structures that are thought to be related to the periseptal annuli that flank the ingrowing septum during cytokinesis. The results show that these structures are localized at potential division sites in non-septate filaments of FtsZ- cells, contrary to previous reports. The positions of the structures along the long axis of the cells in both wild-type cells and FtsZ- filaments were unaffected by the presence of plasmolysis bays at the cell poles. These results do not agree with a previous suggestion that the apparent association of plasmolysis bays with future division sites was artefactual. They support the view that division sites begin to differentiate before the initiation of septal ingrowth and that plasmolysis bays and the annular attachments that define them, mark the locations of these early events in the division process.

Bacterial Proteins↗
PubMed1994

Development of the cell-division site in FtsA- filaments.

Early changes at cell-division sites were studied in non-septate filaments induced by growth of ftsATs mutant cells under non-permissive conditions. The positions of localized regions of plasmolysis were used as markers for the locations of partial and complete annular structures that are thought to be precursors of the periseptal annuli that flank the septum during cytokinesis. The results confirmed that these structures were localized at potential division sites and suggested a model in which older division sites play a role in the generation of new sites for future use, with each older site being used only once for this purpose. The results also suggest that the details of division-site development can profitably be studied in cells in which early events in the differentiation process are uncoupled from the septation event.

Bacterial Adhesion↗
PubMed1992

Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli.

The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that requires the coordinate action of the MinC, MinD, and MinE proteins. In the absence of MinE, the coordinate expression of MinC and MinD leads to a general inhibition of cell division. MinE gives topological specificity to the division inhibition process, so that the septation block is restricted to the cell poles. At normal levels of expression, both MinC and MinD are required for the division block. We show here that, when expressed at high levels, MinC acts as a division inhibitor even in the absence of MinD. The division inhibition that results from MinC overexpression in the absence of MinD is insensitive to the MinE topological specificity factor. The results suggest that MinC is the proximate cause of the septation block and that MinD plays two roles in the MinCDE system--it activates the MinC-dependent division inhibition mechanism and is also required for the sensitivity of the division inhibition system to the MinE topological specificity factor.

Cell Division↗
PubMed1991

A factor that positively regulates cell division by activating transcription of the major cluster of essential cell division genes of Escherichia coli.

Cell division in Escherichia coli requires the products of the ftsQ, ftsA and ftsZ genes. It is not known how the cell regulates the cellular concentrations of these essential elements of the division system. We describe here a factor that activates cell division by specifically increasing transcription from one of the two promoters that lie immediately upstream of the ftsQAZ gene cluster. The trans-acting factor is the product of the sdiA gene, which was isolated on the basis of its ability to suppress the division inhibitory effect of the MinC/MinD division inhibitor. In addition, the sdiA gene product suppressed the action of other chromosomally encoded division inhibitors, induced minicell formation in wild type cells, and restored division activity to an ftsZ temperature-sensitive mutant grown under nonpermissive conditions. All of these properties were explained by the ability of the sdiA gene product specifically to increase transcription of the ftsQAZ gene cluster, resulting in an increase in cellular concentration of the FtsZ protein. The sdiA gene product is the first factor thus far identified that specifically regulates expression of this key group of cell division genes.

Amino Acid Sequence↗