William R. Cook

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.

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 ftsZ null 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.

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.

Biogenesis of cell division sites in ftsA and ftsZ filaments

The development of nascent cell division sites was studied in Escherichia coli strains containing ftsAts and ftsZts mutations in which septal development is arrested after shift to the restrictive temperature. Division sites were studied by measuring positions of plasmolysis bays, a visible marker for periseptal annuli. Annuli are circumferential zones of membrane adhesion which represent the earliest known structural differentiation at developing septal sites. Two patterns of annulus development were observed in the mutant strains. Normal numbers of new annuli were generated in filaments of both mutants, but in ftsZ filaments annuli failed to mature and to become properly localized, suggesting that ftsZ gene product is first required for maturation of annuli. In contrast, mature annuli accumulated at division sites in ftsA filaments, suggesting that the ftsA gene product is required at a stage after maturation and localization of nascent annuli.

Isolation of rat liver microsomal short-chain β-ketoacyl-coenzyme A reductase and trans-2-enoyl-coenzyme A hydratase: evidence for more than one hydratase

An enzyme preparation (IIIB) isolated from liver microsomes of untreated male rats was found to contain two activities--short-chain trans-2-enoyl-CoA hydratase and beta-ketoacyl-CoA reductase. The hydratase was purified more than 1000-fold, while the reductase activity was purified over 600-fold. Employing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a single band with a molecular weight of 76,000 was observed. Although attempts to separate these two activities have failed, it remains to be established whether the final preparation contains a single enzyme with two activities or two separate enzymes. The hydratase was most active toward crotonyl-CoA, followed by trans-2-hexenoyl-CoA (6:1) and -octenoyl-CoA (8:1); the enzyme was essentially inactive toward substrates containing more than eight carbon atoms. The Vmax for crotonyl-CoA was 2117 mumol/min/mg protein, while the Km was 59 microM. Using acetoacetyl-CoA as substrate, the Vmax for the beta-ketoacyl-CoA reductase was over 60 mumol/min/mg protein and the Km was 37 microM; the Vmax for beta-ketopalmitoyl-CoA was only 15% of that observed with acetoacetyl-CoA, although the Km was 6 microM. During the course of purification, a second short-chain hydratase was discovered (fraction IVA); unlike IIIB, this fraction catalyzed the hydration of 4:1, 6:1, and 8:1 at similar rates. The partially purified preparation yielded maximal activity with 8:1 CoA (apparent Vmax 35 mumol/min/mg), followed by 6:1 CoA, 4:1 CoA, and 10:1 CoA; longer chain CoAs were relatively poor substrates, with trans-2-hexadecenoyl CoA about 0.1 as active as 8:1 CoA. On SDS-gels, fraction IVA contained four bands, all of which were below 60,000 Mr. Proteases, such as trypsin, chymotrypsin, and subtilisin, were found to completely inactivate both enzyme fractions.