Duplication of Escherichia coli during inhibition of net phospholipid synthesis.

In Escherichia coli BB26-36, the inhibition of net phospholipid synthesis during glycerol starvation affected cell duplication in a manner that was similar in some respects to that observed during the inhibition of protein synthesis. Ongoing rounds of chromosome replication continued, and cells in the D period divided. The initiation of new rounds of chromosome replication and division of cells in the C period were inhibited. Unlike the inhibition of protein synthesis, however, the accumulation of initiation potential in dnaA and dnaC mutants at the nonpermissive temperature was not affected by the inhibition of phospholipid synthesis. Furthermore, proteins synthesized during the inhibition of phospholipid synthesis can be utilized later for division. The results are consistent with a dual requirement for protein and phospholipid synthesis for both the inauguration of new rounds of chromosome replication and the initiation of septum formation. Once initiated, both processes progress to completion independent of continuous phospholipid and protein synthesis.     

Differential membrane phospholipid synthesis during the cell cycle of Caulobacter crescentus.

The pattern of phospholipid synthesis during the cell cycle of Caulobacter crescentus has been determined. Although the phospholipid composition of swarmer and stalked cells was indistinguishable in continuously labeled cultures if the two cell types were pulse-labeled for a short time period, marked differences in the pattern of phospholipid synthesis were detected. Pulse-labeled swarmer cells exhibited a higher proportion of phosphatidic acid and a lower proportion of phosphatidylglycerol. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. Stalked cells that developed directly from swarmer cells showed that same phospholipid profile as the swarmer cells. The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis.     

Lipid synthesis in callus and protoplasts during culture: Analysis of dividing protoplasts of Petunia hybrida and non-dividing protoplasts of Parthenocissus tricuspidata

The purpose of the investigation was to study the process, by which protoplasts derived from callus tissue overcome the hydrolysis of membrane phospholipids, which is induced by the isolation. Dividing protoplasts from Petunia hybrida L. (line SFla) and non-dividing protoplasts from Parthenocissus tricurpidara L. crown gall (Morel strain) were fed with sodium [1-¹⁴C]-acetate for 1 h. The incorporation was carried out at protoplast isolation and after 1, 2, 4 and 7 days of culture. The rates of fatty acid and lipid synthesis were then followed. Isolation stress induced diversions in the utilization of the precursor via triacylglycerol ( Petuunia ) or diacylglycerol ( Partheno-cissus ) and a decrease in the synthesis of oleic acid ( Parrhenocissus ). During the first 2 days of culture, the rate of phospholipid synthesis was similar in both systems. Later on, an increase in the rate and in the efficiency of phospholipid synthesis was observed in dividing Petunia protoplasts. The accelerated rate of lipid synthesis allowed them to recover the same partitioning and content in membrane phospholipids as the reference cells in the callus. This process seemed to bc related to cell division, since it was not observed in the non-dividing protoplasts of Parthenocissus .     

Effect of macromolecular synthesis and lytic capacity on surface growth of Streptococcus faecalis.

Exposure of exponential-phase cultures of Streptococcus faecalis to any of three inhibitors of protein synthesis was accompanied by an increase in the average distance that the cross wall extended into the cytoplasm. This resulted in: (i) an increase in the average surface area of the cross wall (Sa) and (ii) septation occurring in the envelope growth sites that were much smaller than the controls. However, although at the concentrations used, all three antibiotics inhibited protein synthesis and autolytic capacity to the same extent and with the same kinetics, cells treated with these agents showed large differences in the rate at which Sa values increased above those of the untreated cells. The largest increases in Sa were observed in cells that synthesized the least amount of cytoplasmic macromolecules (deoxyribonucleic acid, plus ribonucleic acid, plus protein). The observations were interpreted in terms of a model in which a decreased lytic capacity reduces the rate of splitting of the nascent cross wall into two layers of peripheral wall, preferentially using wall precursors to close open cross walls. However, the extent to which centripetal growth occurs would be inversely related to the rate at which cytoplasmic macromolecules are synthesized. In contrast, inhibition of deoxyribonucleic acid synthesis was accompanied by decreased extension of the leading edge of the cross wall into the cytoplasm, thus antagonizing septation. These findings are discussed in relation to the normal cell division cycle of S. faecalis.     

Effect of cycloheximide on lipid metabolism in cells, nuclei and subcellular fractions in the rat liver

Well coordinated stages of inhibition, restoration and stimulation of protein, DNA and RNA synthesis were observed after administration of cycloheximide (3 mg/kg). The changes in lipid synthesis and composition in the nuclei and intranuclear structures were studied at different stages of cycloheximide action. The accumulation and stimulation of lipid synthesis in the nuclei during the inhibition and restoration of protein and DNA syntheses were followed by electron microscopy and labeled precursors methods. Dramatic changes were observed in the phospholipid composition of chromatin and nuclear matrix. The accumulation of minor phospholipid fractions in intranuclear structures was observed during DNA synthesis. The sphingomyelin concentration was predominant and commensurable with those of phosphatidylcholine and phosphatidylethanolamine.     

Rate and topography of cell wall synthesis during the division cycle of Salmonella typhimurium.

The rates of synthesis of peptidoglycan and protein during the division cycle of Salmonella typhimurium have been measured by using the membrane elution technique and differentially labeled diaminopimelic acid and leucine. The cells were labeled during unperturbed exponential growth and then bound to a nitrocellulose membrane by filtration. Newborn cells were eluted from the membrane with fresh medium. The radioactivity in the newborn cells in successive fractions was determined. As the cells are eluted from the membrane as a function of their cell cycle age at the time of labeling, the rate of incorporation of the different radioactive compounds as a function of cell cycle age can be determined. During the first part of the division cycle, the ratio of the rates of protein and peptidoglycan synthesis was constant. During the latter part of the division cycle, there was an increase in the rate of peptidoglycan synthesis relative to the rate of protein synthesis. These results support a simple, bipartite model of cell surface increase in rod-shaped cells. Before the start of constriction, the cell surface increased only by cylindrical extension. After cell constriction started, the cell surface increased by both cylinder and pole growth. The increase in surface area was partitioned between the cylinder and the pole so that the volume of the cell increased exponentially. No variation in cell density occurred because the increase in surface allowed a continuous exponential increase in cell volume that accommodated the exponential increase in cell mass. Protein was synthesized exponentially during the division cycle. The rate of cell surface increase was described by a complex equation which is neither linear nor exponential.     

Division Cycle of Myxococcus xanthus III. Kinetics of Cell Growth and Protein Synthesis

The kinetics of cell growth and protein synthesis during the division cycle of Myxococcus xanthus was determined. The distribution of cell size for both septated and nonseptated bacteria was obtained by direct measurement of the lengths of 8,000 cells. The Collins-Richmond equation was modified to consider bacterial growth in two phases: growth and division. From the derived equation, the growth rate of individual cells was computed as a function of size. Nondividing cells (growth phase) comprised 91% of the population and took up 87% of the time of the division cycle. The absolute and specific growth rates of nondividing cells were observed to increase continually throughout the growth phase; the growth rate of dividing cells could not be determined accurately by this technique because of changes in the geometry of cells between the time of septation and physical separation. The rate of protein synthesis during the division cycle was measured by pulselabeling an exponential-phase culture with radio-active valine or arginine and then preparing the cells for quantitative autoradiography. By measuring the size of individual cells as well as the number of grains, the rate of protein synthesis as a function of cell size was obtained. Nondividing cells showed an increase in both the absolute and specific rates of protein synthesis throughout the growth phase; the specific rate of protein synthesis for dividing cells was low when compared to growthphase cells. Cell growth and protein synthesis are compared to the previously reported kinetics of deoxyribonucleic acid and ribonucleic acid synthesis during the division cycle.     

DNA replication initiation, doubling of rate of phospholipid synthesis, and cell division in Escherichia coli.

In synchronized culture of Escherichia coli, the specific arrest of phospholipid synthesis (brought about by glycerol starvation in an appropriate mutant) did not affect the rate of ongoing DNA synthesis but prevented the initiation of new rounds. The initiation block did not depend on cell age at the time of glycerol removal, which could be before, during, or after the doubling in the rate of phospholipid synthesis (DROPS) and as little as 10 min before the expected initiation. We conclude that the initiation of DNA replication is not triggered by the preceding DROPS but requires active phospholipid synthesis. Conversely, when DNA replication initiation was specifically blocked in a synchronized culture of a dnaC(Ts) mutant, two additional DROPS were observed, after which phospholipid synthesis continued at a constant rate for at least 60 min. Similarly, when DNA elongation was blocked by thymine starvation of a synchronized culture, one additional DROPS was observed, followed by linear phospholipid accumulation. Control experiments showed that specific inhibition of cell division by ampicillin, heat shock, or induction of the SOS response did not affect phospholipid synthesis, suggesting that the arrest of DROPS observed was due to the DNA replication block. The data are compatible with models in which the DROPS is triggered by an event associated with replication termination or chromosome segregation.     

Phospholipid synthesis during the cell division cycle of Escherichia coli.

Stepwise changes in the rate of phosphatidylethanolamine and phospholipid synthesis during the cell division cycle of Escherichia coli B/r were observed. The cell ages at the increases were found to be a function of the growth rate. At each growth rate, the increase occurred around the time new rounds of chromosome replication were inaugurated in the cycle.     

Growth and cell division of Mycobacterium avium

The rates of cell division and of protein, DNA and RNA synthesis upon transition of Mycobacterium avium to and from rich medium were examined. The changes in cell morphology (elongation) were also examined by optical and electron microscopy. Upon transfer from poor to rich medium, the rate of synthesis of RNA increased rapidly, followed by an increase in protein synthesis within 3 h and by an increase in DNA synthesis within 7 h; cell division began after a lag of about 10 h. Upon transfer from rich to poor medium, the preshift rates for protein and DNA synthesis changed to postshift rates after 3 h and 7 h, respectively; RNA synthesis stopped immediately, there was a transient fall in total RNA, and synthesis was resumed at a new rate only after 24 h. After the period of adjustment to new medium, the bacteria entered the postshift growth in which cell size, the increase in cell mass (absorbance at 650 nm) and viable counts, and the rates of synthesis of protein, DNA and RNA were constant. Ultrastructural examination of elongated cells during the adjustment period showed that they had septa at different stages of formation, but no evidence of fragmentation was found. It was concluded that cell division in M. avium was by binary fission, and that the notion of a life-cycle was not supported by present findings.     

Lipid synthesis during reinitiation of growth from stationary phase cultures of Candida albicans

Lipid synthesis has been studied in the dimorphic fungus Candida albicans. ¹⁴C-acetate incorporation into lipid material was used to measure new lipid synthesis in two cultures in which either yeast or mycelial growth was initiated from stationary phase yeast cells. When resuspended in fresh medium at 37 °C, cells resume growth and change morphology while at 30 °C cells resume budding growth. When resuspended at the appropriate temperature, both yeast and germ tube cultures immediately incorporated ¹⁴C-acetate into lipid material. The labeled lipid was more or less evenly divided between neutral and phospholipid. Phosphatidyl choline was the major phospholipid fraction and along with phosphatidyl ethanolamine accounted for 60–65 % of the total phospholipid. Lipid synthesis during growth initiation of either morphology showed a similar pattern, with no significant differences observed in neutral or phospholipid or phospholipid components between yeast and mycelial forms.     

Morphological effect of cerulenin treatment on Streptococcus faecalis as studied by ultrastructure reconstruction.

Exponential-phase cells of Streptococcus faecalis ATCC 9790 were treated with a concentration of cerulenin (5 micrograms/ml) that has been shown to block both lipoteichoic acid and lipid synthesis and cell division within 10 min. The morphological effect of this treatment was studied by making three-dimensional reconstructions of cells based on measurements taken from axial thin sections. This analysis indicated that cerulenin interferes with cell division by inhibiting normal constriction of the division furrow and centripetal growth of the cross wall in envelope growth sites. Rather than dividing, many of the sites in treated cells apparently continue to elongate and produce abnormally large amounts of peripheral wall surface. These observations were interpreted in terms of a previously proposed model in which cerulenin would prevent the synthesis of a lipid-containing inhibitor of autolytic enzyme activity needed for division. In addition, measurements showed that the average number of envelope growth sites per cell increased during treatment, suggesting that although cerulenin treatment blocks division, it does not interfere with the formation of new envelope growth sites. It was also observed that the size and frequency of mesosomes did not decline during the 60-min period of drug treatment. This tends to decrease the likelihood that mesosomes are formed from a pool of intracellular membrane precursors that would be depleted during a period of restricted lipid biosynthesis.     

Control of membrane lipid synthesis in Escherichia coli during growth and during the stringent response.

The regulation of phospholipid synthesis in cells of Escherichia coli was studied in vivo during growth and during the stringent response to amino acid starvation. Strains harboring the hybrid plasmid pLC44-14 (Clark, L., and Carbon, J. (1976) Cell 9, 91-99), which had increased levels of glycerophosphate acyltransferase, were used to study the involvement of this enzyme in the control of phospholipid synthesis. In addition, regulation was studied by measuring the levels of three early intermediates of phospholipid synthesis:phosphatidic acid, CDP-diglyceride, and dCDP-diglyceride. The liponucleotides were measured by a new enzymatic method which allows determinations to be made on crude lipid extracts. Results from experiments on growing cells are consistent with regulation of membrane lipid synthesis occurring in fatty acid synthesis or at the level of glycerophosphate acylation, but not at any later step. Experiments on the inhibition of lipid synthesis during the stringent response make it possible to rule out explanations which involve the inhibition of a single enzyme; enzymes both before and after the liponucleotides in phospholipid synthesis must be affected.     

Heat shock-induced changes in lipid and protein metabolism in the endoplasmic reticulum of barley aleurone layers

Heat shock in barley aleurone layers induces heat shock protein synthesis and suppresses secretory protein synthesis by selectively destabilizing their mRNAs. In addition, the endoplasmic reticulum (ER) membranes upon which secretory protein mRNAs are translated become vesiculated during heat shock, leading to the hypothesis that ER dissociation and targeted mRNA destabilization are linked mechanistically. Supporting this, ER can be heat adapted, and heat-adapted ER has higher levels of fatty acid saturation in membrane phospholipids which do not vesiculate upon heat shock. Secretory protein mRNAs are also more stable in heat-adapted cells. To understand better heat shock-induced changes in ER membranes, we examined ER membrane proteins and enzymes involved in phosphatidylcholine biosynthesis and phospholipid turnover in heat-shocked aleurone cells. Heat shock significantly increased the activity of phospholipases A2 and D, and shortly thereafter significant but gradual increases in choline kinase and phosphocholine glyceride transferase activities and a sharp increase in phosphorylcholine citidyl transferase activity were observed. Only minor changes were observed in SDS-PAGE analyses of proteins from sonicated ER membranes fractionated on continuous sucrose gradients. Overall, heat shock reduced total lipid in ER membranes relative to protein, and in intact, ultracentrifuged aleurone cells examined by light and electron microscopy the ER band appeared to increase in density. The changes in phospholipid metabolism coupled with the suppression of secretory protein synthesis indicate that in addition to inducing a classic heat shock response, high temperature also induces a classic unfolded protein response in the ER of this secretory cell.     

A block in endoplasmic reticulum-to-Golgi trafficking inhibits phospholipid synthesis and induces neutral lipid accumulation

Seeking to better understand how membrane trafficking is coordinated with phospholipid synthesis in yeast, we investigated lipid synthesis in several Sec(-) temperature-sensitive mutants, including sec13-1. Upon shift of sec13-1 cells to the restrictive temperature of 37 degrees C, phospholipid synthesis decreased dramatically relative to the wild type control, whereas synthesis of neutral lipids, especially triacylglycerol (TAG), increased. When examined by fluorescence microscopy, the number of lipid droplets appeared to increase and formed aggregates in sec13-1 cells shifted to 37 degrees C. Electron microscopy confirmed the increase in lipid droplet number and revealed that many were associated with the vacuole. Analysis of lipid metabolism in strains lacking TAG synthase genes demonstrated that the activities of the products of these genes contribute to accumulation of TAG in sec13-1 cells after the shift to 37 degrees C. Furthermore, the permissive temperature for growth of the sec13-1 strain lacking TAG synthase genes was 3 degrees C lower than sec13-1 on several different growth media, indicating that the synthesis of TAG has physiological significance under conditions of secretory stress. Together these results suggest that following a block in membrane trafficking, yeast cells channel lipid metabolism from phospholipid synthesis into synthesis of TAG and other neutral lipids to form lipid droplets. We conclude that this metabolic switch provides a degree of protection to cells during secretory stress.     

Envelope protein synthesis and inhibition of cell division in Escherichia coli during inactivation of the B subunit of DNA gyrase

The rates of synthesis of inner and outer membrane proteins of Escherichia coli K12 during inhibition of cell division have been studied. When cell division was inhibited, either by treatment of wild-type cells with the antibiotic clorobiocin (an inhibitor of the B subunit of DNA gyrase) or by a temperature shift of a gyrB-ts mutant, a 40% reduction in the rate of synthesis of total outer membrane protein relative to that of the inner membrane was observed. When a gyrB-ts mutant was shifted to high temperature under conditions which allowed continued cell division, this large reduction in the rate of synthesis of outer membrane protein relative to inner membrane protein was not observed. In contrast to the results obtained with clorobiocin, inhibition of cell division by the beta-lactam antibiotic cefuroxime did not cause any detectable disturbance in the rate of synthesis of either inner or outer membrane protein. This demonstrates that inhibition of septum formation per se does not perturb synthesis of envelope protein. The data obtained are consistent with a model in which the rate of synthesis and therefore expansion of outer membrane is one of many conditions which must be satisfied before septum formation can occur. The results are discussed in relation to such a model, and to previous findings which have shown that the rate of synthesis of outer membrane proteins displays a linear mode with an abrupt doubling in rate at a discrete point in the cell cycle.     

The incorporation of [1-14C]acetate into lipids during embryogenesis in oil palm tissue cultures

A tissue culture line of oil palm produced embryoids and during the embryogenesis large quantities of lipid were stored in the cells. The synthesis of the lipid was monitored by measuring incorporation of[1-¹⁴C]acetate, under optimum conditions, into the total lipid and separation by TLC into neutral and polar lipid. Both synthesis of triacylglycerol and polar lipid increased during embryoid formation. A rapid increase in the formation of polar lipid occurred in the period just before the embryoids became visible and this probably corresponded with the increased rate of cell division that occurred at that time.     

Lipid spirals in Bacillus subtilis and their role in cell division

The fluid mosaic model of membrane structure has been revised in recent years as it has become evident that domains of different lipid composition are present in eukaryotic and prokaryotic cells. Using membrane binding fluorescent dyes, we demonstrate the presence of lipid spirals extending along the long axis of cells of the rod-shaped bacterium Bacillus subtilis. These spiral structures are absent from cells in which the synthesis of phosphatidylglycerol is disrupted, suggesting an enrichment in anionic phospholipids. Green fluorescent protein fusions of the cell division protein MinD also form spiral structures and these were shown by fluorescence resonance energy transfer to be coincident with the lipid spirals. These data indicate a higher level of membrane lipid organization than previously observed and a primary role for lipid spirals in determining the site of cell division in bacterial cells.     

The inhibition of phospholipid synthesis in escherichia coli by phenethyl alcohol.

The kinetics of lipid metabolism during phenethyl alcohol treatment of Escherichia coli were examined. Phenethyl alcohol at a non-bacteriostatic concentration reduces the accumulation of [32-P] phosphate into phospholipids and alters the phospholipid composition of the cell membrane. The changes in phospholipid composition are a result of the inhibitory effect of phenethyl alcohol on the rates of synthesis of the individual phospholipids. The inhibition in the rate of phosphatidylethanolamine synthesis by phenethyl alcohol was twice the inhibition in the rate of phosphatidyglycerol synthesis. The de novo rate of cardiolipin synthesis was only slightly inhibited. However, net cardiolipin accumulation increased during phenethyl alcohol treatment due to a more rapid turnover of phosphatidylglycerol to cardiolipin. Phenethyl alcohol also altered the fatty acid composition of the cell as a result of its inhibitory effect on the rate of individual fatty acid synthesis. However, the inhibition of phospholipid synthesis was not reversed by fatty acid supplementation of phenethyl alcohol treated cells. This result indicates that phenethyl alcohol does not inhibit phospholipid synthesis solely at the level of fatty acid synthesis.     

Lipid Synthesis and Abundance of Acetyl CoA Carboxylase in Isochrysis galbana (Prymnesiophyceae) Following Nitrogen Starvation

Fatty acid content and the rate of lipid synthesis were measuredin the marine prymnesiophyte Isochrysis galbana grown undernitrogen starvation and in cultures recovering from nitrogendeprivation. Nitrogen starvation imposed a reduction in cellularsoluble protein content, variation in fatty acid compositionand reduction in the in vitro activity of the enzyme acetylCoA carboxylase. An increase in total fatty acid content isattributed to a differential reduction in cell division andthe rate of lipid synthesis. Recovery from nitrogen deprivationwas characterized by an increase in cellular soluble proteincontent and in the rate of lipid synthesis. Although the invitro activity of acetyl CoA carboxylase increased as the culturesrecovered from nitrogen starvation, the total cellular fattyacid content decreased, evidently due to an acceleration incell division. The relative cellular pool size of acetyl CoAcarboxylase determined by immunoblotting decreased under nitrogenstarvation conditions and increased as cells recovered fromit. Cellular accumulation of acetyl CoA carboxylase during recoveryfrom nitrogen starvation is ascribed to de novo synthesis ofthe enzyme that takes place in the cytoplasm. However, photosyntheticproteins such as ribulose bisphosphate carboxylase are synthesizedearlier than acetyl CoA carboxylase in the recovery process. (Received June 12, 1992; Accepted September 21, 1992)