Rate and topography of peptidoglycan synthesis during cell division in Escherichia coli: concept of a leading edge

Abstract

The rate at which the peptidoglycan of Escherichia coli is synthesized during the division cycle was studied with two methods. One method involved synchronization of E. coli MC4100 lysA cultures by centrifugal elutriation and subsequent pulse-labeling of the synchronously growing cultures with [meso-3H] stricted to the site of constriction, whereas in nondividing cells, incorporation was diffuse over the cell envelope (4, 18, 24). Furthermore, their results indicated that the rate of incorporation increased continuously during the division cycle, with a slight acceleration at the beginning of constriction. On the other hand, Cooper (5) presented a model for Salmonella typhimurium in which there is no such discontinuity in the rate of peptidoglycan synthesis during the division cycle. In that model, the rate of peptidoglycan synthesis is proportional to the rate of protein synthesis during elongation, whereas during constriction, the rate of peptidoglycan synthesis increases relative to the rate of protein synthesis.In this paper, we report on experiments in which the rate and topography of peptidoglycan synthesis was studied with two independent methods, i.e., pulse-labeling of synchronized cultures and autoradiography of pulsed cells. In par-* Corresponding author. ticular, we investigated whether the localized Dap incorporation at the constriction site can be further differentiated, i.e., does the topography and rate of Dap incorporation at the constriction site change during the constriction process (5)? The latter question was studied on the one hand by comparing the topography of Dap incorporation in slightly, moderately, and deeply constricted cells and on the other hand by autoradiography of cells with impaired penicillinbinding protein 3 (PBP3).We found a more or less exponentially increasing rate of peptidoglycan synthesis in pulse-labeled synchronized cells; in addition, by autoradiography, we found a slight acceleration in this rate during constriction. The topography of Dap incorporation after the switch from diffuse incorporation into the lateral walls to increased incorporation at the constriction site revealed that Dap incorporation occurred predominantly at the leading edge of the constriction site. The results also indicated that the leading edge had a constant activity during the constriction process. On the basis of autoradiography of cells with impaired PBP3, the constriction process could be subdivided into a PBP3-independent initiation and a subsequent PBP3-dependent completion of constriction.MATERIALS AND METHODS Strains and media. The E. coli K-12 strain MC4100 lysA was used (27), and in some experiments, a pbpB derivative of it (22) was used. MC4100 lysA was grown at 37°C in minimal citrate medium with glucose as the carbon source unless stated otherwise. The medium was supplemented with 50 Lg of lysine, methionine, and threonine per ml (27). The mass doubling time was ca. 45 min. The pbpB strain, carrying a temperature-sensitive mutation in the gene for PBP3c, was grown in the same med...

Defining the rate-limiting processes of bacterial cytokinesis

“…Thus, our measurements indicate that, during constriction, cell elongation occurs predominately through septal cell wall growth, because substantial contributions from lateral growth along the cell body would result in v c /v ec < 1. This finding is consistent with early observations that PG incorporation activity is concentrated at the septum during the constriction period (89,90) and highlights the method's precision in determining the elongation and septum closure rates. Collectively, these correlations suggest that the differences in septum closure rate observed in different wt strains under varying growth conditions can be attributed to differences in PG synthesis rates and that the cellular processes limiting cell wall synthesis may also limit the rate of septum closure.…”

“…This lack of a unifying, FtsZ-centric theme prompted us to explore the roles of other divisome components. We first examined the role of cell wall PG synthesis activity, which takes place in two modes during the cell cycle: initially along the lateral cell body and then predominantly septum-localized during constriction (89,90). Septal cell wall synthesis is directed by the divisome, and lateral cell wall synthesis is directed by the 'elongasome' (91).…”

Patchiness of murein insertion into the sidewall of Escherichia coli

“…The work presented here also extends the findings of Schwarz et al (1975), Woldringh et al (1987), Wientjes & Nanninga (1989) and De Pedro et al (1997) in showing that the region destined for pole formation is formed of entirely new material and is not an admixture of old and new murein. In a chase of one and two doubling times, there are regions of new (fully non-labelled) material inserted in the cylindrical sidewalls.…”

Autoradiographic analysis of diaminopimelic acid incorporation in filamentous cells of Escherichia coli: repression of peptidoglycan synthesis around the nucleoid

“…Constriction between segregated nucleoids, as occurs in wild-type cells and in recovering cell division mutants, is accompanied by a local increase in PG synthesis rate at the constriction site (15,17,19). Coordinately with this local increase, the synthesis rate in the lateral wall decreases (1,10,17).…”