Complete Engulfment Research Articles

Expression of spoIIIJ in the prespore is sufficient for activation of sigma G and for sporulation in Bacillus subtilis.

During sporulation in Bacillus subtilis, the prespore-specific developmental program is initiated soon after asymmetric division of the sporangium by the compartment-specific activation of RNA polymerase sigma factor sigma(F). sigma(F) directs transcription of spoIIIG, encoding the late forespore-specific regulator sigma(G). Following synthesis, sigma(G) is initially kept in an inactive form, presumably because it is bound to the SpoIIAB anti-sigma factor. Activation of sigma(G) occurs only after the complete engulfment of the prespore by the mother cell. Mutations in spoIIIJ arrest sporulation soon after conclusion of the engulfment process and prevent activation of sigma(G). Here we show that sigma(G) accumulates but is mostly inactive in a spoIIIJ mutant. We also show that expression of the spoIIIGE155K allele, encoding a form of sigma(G) that is not efficiently bound by SpoIIAB in vitro, restores sigma(G)-directed gene expression to a spoIIIJ mutant. Expression of spoIIIJ occurs during vegetative growth. However, we show that expression of spoIIIJ in the prespore is sufficient for sigma(G) activation and for sporulation. Mutations in the mother cell-specific spoIIIA locus are known to arrest sporulation just after completion of the engulfment process. Previous work has also shown that sigma(G) accumulates in an inactive form in spoIIIA mutants and that the need for spoIIIA expression for sigma(G) activation can be circumvented by the spoIIIGE155K allele. However, in contrast to the case for spoIIIJ, we show that expression of spoIIIA in the prespore does not support efficient sporulation. The results suggest that the activation of sigma(G) at the end of the engulfment process involves the action of spoIIIA from the mother cell and of spoIIIJ from the prespore.

A dispensable role for forespore-specific gene expression in engulfment of the forespore during sporulation of Bacillus subtilis.

During the stage of engulfment in the Bacillus subtilis spore formation pathway, the larger mother cell engulfs the smaller forespore. We have tested the role of forespore-specific gene expression in engulfment using two separate approaches. First, using an assay that unambiguously detects sporangia that have completed engulfment, we found that a mutant lacking the only forespore-expressed engulfment protein identified thus far, SpoIIQ, is able to efficiently complete engulfment under certain sporulation conditions. However, we have found that the mutant is defective, under all conditions, in the expression of the late-forespore-specific transcription factor sigma(G); thus, SpoIIQ is essential for spore production. Second, to determine if engulfment could proceed in the absence of forespore-specific gene expression, we made use of a strain in which activation of the mother cell-specific sigma factor sigma(E) was uncoupled from forespore-specific gene expression. Remarkably, engulfment occurred in the complete absence of sigma(F)-directed gene expression under the same conditions permissive for engulfment in the absence of SpoIIQ. Our results demonstrate that forespore-specific gene expression is not essential for engulfment, suggesting that the machinery used to move the membranes around the forespore is within the mother cell.

SpoIIB localizes to active sites of septal biogenesis and spatially regulates septal thinning during engulfment in bacillus subtilis.

A key step in the Bacillus subtilis spore formation pathway is the engulfment of the forespore by the mother cell, a phagocytosis-like process normally accompanied by the loss of peptidoglycan within the sporulation septum. We have reinvestigated the role of SpoIIB in engulfment by using the fluorescent membrane stain FM 4-64 and deconvolution microscopy. We have found that spoIIB mutant sporangia display a transient engulfment defect in which the forespore pushes through the septum and bulges into the mother cell, similar to the situation in spoIID, spoIIM, and spoIIP mutants. However, unlike the sporangia of those three mutants, spoIIB mutant sporangia are able to complete engulfment; indeed, by time-lapse microscopy, sporangia with prominent bulges were found to complete engulfment. Electron micrographs showed that in spoIIB mutant sporangia the dissolution of septal peptidoglycan is delayed and spatially unregulated and that the engulfing membranes migrate around the remaining septal peptidoglycan. These results demonstrate that mother cell membranes will move around septal peptidoglycan that has not been completely degraded and suggest that SpoIIB facilitates the rapid and spatially regulated dissolution of septal peptidoglycan. In keeping with this proposal, a SpoIIB-myc fusion protein localized to the sporulation septum during its biogenesis, discriminating between the site of active septal biogenesis and the unused potential division site within the same cell.

Free motion of a cylinder moving below and through a free surface

This paper presents the fully non-linear inviscid-flow calculations of the deformations of an initially calm free surface caused by the free motion of a horizontal cylinder starting from rest from a prescribed depth. The following cases are studied: (a) Upwards motion of a cylinder of specific gravity M b<1. (b) Downwards motion of a cylinder with M b>1. (c) Motion of a cylinder with M b=1, started with an impulsive horizontal velocity. (d) Downwards motion of a cylinder with M b>1, initially half-submerged. The complete engulfment of the cylinder, so that it changes from being surface-piercing to being totally submerged, requires special numerical treatment.

Evidence that the spoIIM gene of Bacillus subtilis is transcribed by RNA polymerase associated with sigma E

We have investigated the temporal and spatial regulation of spoIIM, a gene of Bacillus subtilis whose product is required for complete septum migration and engulfment of the forespore compartment during sporulation. The spoIIM promoter was found to become active about 2 h after the initiation of sporulation. The effects of mutations on the expression of a spoIIM-lacZ fusion were most consistent with its utilization by sigma-E-associated RNA polymerase (E sigma E). A unique 5' end of the in vivo spoIIM transcript was detected by primer extension analysis and was determined to initiate at the appropriate distance from a sequence conforming very closely to the consensus for genes transcribed by E sigma E. A partially purified preparation of E sigma E produced a transcript in vitro that initiated at the same nucleotide as the primer extension product generated from in vivo RNA. Ectopic induction of sigma E synthesis during growth resulted in the immediate and strong expression of a spoIIM-lacZ fusion, but an identical fusion was completely unresponsive to induced synthesis of either sigma F or sigma G under similar conditions. The results of plasmid integration-excision experiments in which the spoIIM gene was reversibly disrupted by a temperature-sensitive integrational vector suggested that spoIIM expression is required in the forespore compartment, but direct examination of subcellular fractions enriched for mother cell or forespore material indicated that spoIIM expression cannot be confined to the forespore. We conclude that spoIIM is a member of the sigma E regulon and that it may be transcribed exclusively by E sigma E. We discuss the implications of this conclusion for models in which activation of sigma E in the mother cell is proposed to be a part of the mechanism responsible for initiating separate programs of gene activity in the two sporangium compartments.

Fire engulement of LPG tanks: heatup, a predictive model

A predictive model of the behaviour of horizontal pressurised LPG vessels engulfed by fire is described. Complete fire engulfment is treated with a heat flux distribution that is both variable in the vertical direction and time dependent. Heat flow through the tank walls, convective and radiative exchange to the fluid and heat and mass transfer between the liquid and vapour are incorporated. The liquid and vapour zones are each assumed to be separately well mixed. The operation of different designs of pressure relief valves is simulated. Model validation was against extensive measurements on 0.25, 1 and 5 tonne LPG tanks filled to a range of levels and engulfed in kerosine pool fires. HEATUP successfully predicted pressure relief valve opening times, discharge rates, pressure histories and average liquid and vapour temperatures for the different tank sizes and fill levels. Predicted mean wall temperatures in both liquid and vapour zones also agreed with measurements.

Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin.

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

Configurations of gas-liquid two-phase bubbles in immiscible liquid media

The configuration of a “two-phase bubble” constituted of a gas phase and a liquid phase in an immiscible liquid medium is classified into three types: complete engulfing of a gas bubble inside a liquid shell, partial coalescence of a gas bubble and a liquid drop forming a three-phase contact line, and non-coalescence whereby a gas bubble and a liquid drop remain separated. Simple criteria have been presented by which the favorable type of configuration in a given system is predicted from the values of the spreading coefficients characterizing the system. Experiments using some combinations of liquids as well as air suggest the general validity of the criteria.