Murein biosynthesis during a synchromous cell cycle of Escherichia coli B.

The last stages of murein biosynthesis were studied in relation to the division cycle of Escherichia coli in cells synchronized by amino acid starvation (Ron et al., J. Bacteriol. 123:374--376, 1975). Murein synthesis and the activities of the D-alanine carboxypeptidase and transpeptidase were found to vary significantly during the cell cycle. Maximal synthesis and transpeptidation were observed immediately after cell division, whereas maximal D-alanine carboxypeptidase activity was detected before cell division. These results are in agreement with our earlier findings that before cell division there is a stage of increased hydrolysis of the C-terminal D-alanine moiety of newly synthesized murein strands.     

Production and characterization of a monoclonal antibody to the O-acetylated peptidoglycan of Proteus mirabilis.

A monoclonal antibody (PmPG5-3) specific for the O-acetylated peptidoglycan of Proteus mirabilis 19 was produced by an NS-1 myeloma cell line and purified from ascites fluid by a combination of ammonium sulfate precipitation and affinity chromatography. The monoclonal antibody (an immunoglobulin M) was characterized by a competition enzyme-linked immunosorbent assay to be equally specific for both insoluble and soluble O-acetylated peptidoglycan but weakly recognized chemically de-O-acetylated P. mirabilis peptidoglycan, the non-O-acetylated peptidoglycans from Escherichia coli and Bacillus subtilis, and the peptidoglycan monosaccharide precursors N-acetylglucosamine and N-acetylmuramic acid dipeptide. The monoclonal antibody did not react with D-alanine or lipopolysaccharide isolated from P. mirabilis. Based on this evidence, the binding epitope on the P. mirabilis peptidoglycan is predicted to be linear and to comprise the glycan backbone, including both the N- and O-acetyl moieties. Monoclonal antibody PmPG5-3 was used to localize the O acetylation of the P. mirabilis peptidoglycan by immunoelectron microscopy. Murein sacculi of P. mirabilis were heavily and randomly labelled with the immunogold, whereas very little labelling and no labelling were observed on the sacculi isolated from de-O-acetylated P. mirabilis and E. coli, respectively. Based on the apparent pattern of immunogold labelling, a physiological role for peptidoglycan O acetylation in P. mirabilis is proposed.     

Cell cycle parameters of Proteus mirabilis: interdependence of the biosynthetic cell cycle and the interdivision cycle.

We investigated the time periods of DNA replication, lateral cell wall extension, and septum formation within the cell cycle of Proteus mirabilis. Cells were cultivated under three different conditions, yielding interdivision times of approximately 55, 57, and 160 min, respectively. Synchrony was achieved by sucrose density gradient centrifugation. The time periods were estimated by division inhibition studies with cephalexin, mecillinam, and nalidixic acid. In addition, DNA replication was measured by thymidine incorporation, and murein biosynthesis was measured by incorporation of N-acetylglucosamine into sodium dodecyl sulfate-insoluble murein sacculi. At interdivision times of 55 to 57 min murein biosynthesis for reproduction of a unit cell lasted longer than the interdivision time itself, whereas DNA replication finished within 40 min. Surprisingly, inhibition of DNA replication by nalidixic acid did not inhibit the subsequent cell division but rather the one after that. Because P. mirabilis fails to express several reactions of the recA-dependent SOS functions known from Escherichia coli, the drug allowed us to determine which DNA replication period actually governed which cell division. Taken together, the results indicate that at an interdivision time of 55 to 57 min, the biosynthetic cell cycle of P. mirabilis lasts approximately 120 min. To achieve the observed interdivision time, it is necessary that two subsequent biosynthetic cell cycles be tightly interlocked. The implications of these findings for the regulation of the cell cycle are discussed.     

Alterations in the cell envelope composition of Proteus mirabilis during the development of swarmer cells.

Long, swarming cells of Proteus mirabilis had different proportions of some lipopolysaccharide components when compared to short cells, either agar grown or broth grown. Fluorescence spectrophotometry of antibody binding, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the change was in the proportion of lipopolysaccharide with long O-antigenic sidechains, swarmer lipopolysaccharide relative to short sidechain lipopolysaccharide than the non-swarming cells. The proteins and phospholipids of the envelop remained the same during swarmer development. The results are discussed in relation to the increase in flagella synthesis and permeability to some antibacterial agents during swarmer development.     

Transport of thymidine during the cell cycle in mitotically synchronized CHO cells

The kinetics of total uptake of thymidine into the cell were determined for cells which had been mitotically synchronized, plated into scintillation vials and pulsed with five concentrations of [³H]-thymidine at various times during the cell cycle. From Lineweaver-Burk plots of these rates, V_max and K_m values were determined for the transport of thymidine. The V_max values ranged from a low of 2.0 pmoles/ min/10⁶ cells in mid-G1 to a high of 99.7 in mid-S before a decline in late S and G2. K_m values displayed only a 5-fold range in values.     

Genetic analysis of Proteus mirabilis mutants defective in swarmer cell elongation.

Swarmer cell differentiation is a complex process involving the activity of many gene products. In this report, we characterized the genetic locus of Tn5 insertion in each of 12 mutants defective in swarmer cell elongation. The mutations fell into four categories affecting either flagellar biosynthesis or energetics, lipopolysaccharide and cell wall biosynthesis, cellular division, or proteolysis of peptides.     

Changes in murein composition during the cell cycle of Caulobacter crescentus

The amino acid and muropeptide compositions of murein (peptidoglycan) isolated from populations of Caulobacter crescentus predominantly composed of swarmer or stalked cells were determined and compared with the structure of murein sacculi obtained from a population of unsegregated cells. It appears that in spite of vast morphological alterations in the course of the cell cycle, the murein composition of the various cell types is not markedly different.     

Uptake of amino acids and thymidine during the first cell cycle of synchronized hamster cells.

The net total uptake of four amino acids (valine, leucine, lysine and methionine) used at concentrations required for growth, and of thymidine at tracer concentrations, has been studied during the first cell cycle of an asparagine-dependent strain of transformed BHK cells synchronized by asparagine starvation. The rate of the total uptake of the amino acids, the free pool of the amino acids taken up, and the rate of their incorporation into protein at the end of the first cell cycle were, on the average, 12-fold that at the beginning of the cell cycle. The increase in these parameters during the cell cycle was not linear. The uptake of thymidine started before the onset of DNA synthesis and proceeded linearly beyond the peak of the S phase. The rate of accumulation of thymidine into the acid-soluble fraction also increased during the S phase, apart from a tendency to plateau off at the peak of this phase. It reached a second plateau towards the end of the cell cycle, and then declined slightly. Evidence is presented which suggests that the total quantity of protein synthesized during the cell cycle is more than the newly synthesized protein present in the cells at the end of the cell cycle; this indicated degradation and/or secretion of a substantial proportion of the newly synthesized protein. The total protein synthesized at different time points in the cell cycle appeared to contain different proportions of the amino acids used.     

Studies on changes in DNA polymerase activity during the cell cycle in synchronized KB cells.

We have demonstrated the presence of two DNA polymerases in KB cells and studied the variation of their activities in a synchronous cell population. During the cell cycle we observed in nuclei, only one DNA dependent DNA polymerase, the 3.4 S or minipolymerase, and similarly in the cytoplasm only one enzyme, the 8.3 S or maxipolymerase. The former shows preference for native DNA and the latter for denatured DNA. Their Mg++ and K+ requirements are different and their pH optima are 8.5 and 7 for nuclear polymerase and cytoplasmic polymerase respectively. The cytoplasmic polymerase activity remains stable from one cell cycle to the other with each cell reconstituting its stock at the start of the following cycle (G1 and early S phases). On the contrary nuclear activity decreases in G2, M and early G1, then increases to a maximum in the middle of the S phase. This fluctuation in enzyme activity could be due to degradation, transfer to the cytoplasm or the association of the enzyme with the chromatin and/or the nuclear membrane after completion of DNA synthesis. Our results do not permit us to choose between these three hypotheses. However their significance is discussed in the light of the results obtained by some authors who, on the contrary, have tended to minimise the role of the minipolymerase in DNA duplication, whereas we, from our findings, ascribe a preponderant role to this enzyme. The cytoplasmic maxipolymerase (8.3 S) may simply be a storage form of the enzyme from which minipolymerase can be formed as needed.     

Uptake of amino acids and thymidine during the first cell cycle of synchronized hamster cells

Summary The net total uptake of four amino acids (valine, leucine, lysine and methionine) used at concentrations required for growth, and of thymidine at tracer concentrations, has been studied during the first cell cycle of an asparagine-dependent strain of transformed BHK cells synchronized by asparagine starvation. The rate of the total uptake of the amino acids, the free pool of the amino acids taken up, and the rate of their incorporation into protein at the cell cycle. The increase in these parameters during the cell cycle was not linear. The uptake of thymidine started before the onset of DNA synthesis and proceeded linearly beyond the peak of the S phase. The rate of accumulation of thymidine into the acid-soluble fraction also increased during the S phase, apart from a tendency to plateau off at the peak of this phase. It reached a second plateau towards the end of the cell cycle, and then declined slightly. Evidence is presented which suggests that the total quantity of protein synthesized during the cell cycle is more than the newly synthesized protein present in the cells at the end of the cell cycle; this indicates degradation and/or secretion of a substantial proportion of the newly synthesized protein. The total protein synthesized at different time points in the cell cycle appeared to contain different proportions of the amino acids used.     

Murein and lipopolysaccharide biosynthesis in synchronized cells of Escherichia coli K 12 and the effect of penicillin G,mecillinam and nalidixic acid

The incorporation of radioactive N-acetylglucosamine into murein and lipopolysaccharide of synchronized cells of Escherichia coli K 12 was followed over 100 min in the presence of antibiotics. At 20 min intervals cell walls were prepared. Lipopolysaccharide and murein sacculi were isolated and the radioactivity was quantified in both polymers. Labelled, newly synthesized murein was characterized according to murein subunits linked to lipoprotein, and the degree of crosslinkage. Furthermore, murein subunits containing anhydromuramic acid were determined, permitting the calculation of the average glycan chain length. The results indicated that penicillin G at 30 g/ml stimulated the incorporation of new murein subunits into sacculi followed by a sudden increase in lipopolysaccharide incorporation into the outer membrane. The degree of crosslinkage in murein synthesized in the presence of 30 g/ml penicillin G was higher than in the control, and almost twice as high as in murein synthesized in the presence of 20 g/ml nalidixic acid. Both antibiotics inhibited cell division at the concentrations indicated. Murein synthesized in the presence of 2 g/ml mecillinam also showed higher crosslinkage. However, about twice as much anhydromuramic acid-containing subunits were observed as in the control. At the same time lipopolysaccharide incorporation into the outer membrane was stimulated two- to three-fold.Abbreviation GlcNAc N-acetylglucosamine     

Ultrastructural characteristics of Proteus vulgaris and Proteus mirabilis cells differing in the capacity for swarming

Specific differences in the structure of colonies and the location of microbial cells in colonies, characteristic for aggregating and nonaggregating genetically related pairs of P. vulgaris and P. mirabilis strains, have been demonstrated by means of transmission and scanning electron microscopy. In calculating the number of flagellae per 100 outlines of microbial bodies revealed in negatively stained preparations, the fact that both aggregating and nonaggregating bacteria possess practically the same number of flagellae, on the average 4-8 flagellae per microbial cell outline, has been established. This fact indicates that the presence of flagellae in microbial cells is unrelated to their capacity for swarming.     

Expression of proliferation associated antigens in the cell cycle of synchronized mammalian cells.

Flow cytometric bivariate analysis was used to investigate the expression of PCNA, p120 and p145 during the cell cycle of a mammalian cell line (CHO-K1). Initially, aliquots of cells in exponential and plateau (G0) phase were analyzed for proliferation associated antigen expression. Expression of PCNA and p145 during G0 was markedly depressed (less than 12% positive) while 54% of the G0 cells stained positive for p120. The fluorescent intensity (mean channel fluorescence) of these G0 positive p120 cells, however, was only slightly above the mean channel fluorescence (MCF) of cells stained with a negative isotype control. In asynchronous cultures, all three antigens were expressed in greater than 70% of the cells, with PCNA staining being greater than 95%. Cells were then synchronized using mitotic selection (mitotic index of 97%) and antigen levels were measured as cells progressed synchronously through the cell cycle. From DNA analysis histograms, it appeared that the degree of synchrony was approximately 90% throughout the remainder of the cell cycle. The bivariate DNA/PCNA, DNA/p120, and DNA/p145 histograms for mitotic cells indicated that both p120 and p145 expression were elevated (percent positive and MCF) while PCNA levels were near controls (MCF). In early G1, all three markers were depressed (less than 12% positive); however PCNA levels rose precipitously in mid-G1 (greater than 50% positive). In late G1 to early S, p145 levels increased concomitantly with increases in p120. All three antigens were elevated throughout S phase and began to decline as cells moved from G2/M to G1 of the next cell cycle with p145 expression decreasing first. This report indicates that all three proliferation associated antigens studied are differentially expressed in the cell cycle and therefore may be useful in detecting and assessing the proliferation state.     

Activity of three murein hydrolases during the cell division cycle of Escherichia coli K-12 as measured in toluene-treated cells.

The specific activities of three murein hydrolases, carboxypeptidase I, carboxypeptidase II, and amidase were studied with respect to cell division in toluene-treated cells of Escherichia coli K-12. Carboxypeptidase I and amidase activities were constant throughout the division cycle in cells of D11/lac+pro+. Detectable carboxypeptidase II activity varied and was highest at the time of division by a factor of three. Carboxypeptidase II specific activity was also correlated with cell division in BUG 6, a temperature-sensitive mutant (J.N Reeve, D.J. Groves, and D.J. Clark, 1970). Fifteen minutes after shifting BUG 6 from 42 C (nondividing conditions) to 32 C (dividing conditions), there was a rapid resumption of cell division, accompanied by a 10-fold increase in the specific activity of carboxypeptidase II. These results demonstrate a correlation between detectable carboxypeptidase II activity and cell division as reflected by activity in toluene-treated cells. The subcellular location of carboxypeptidase II, a soluble enzyme was found to be periplasmic since it was released by tris(hydroxymethyl)-aminomethane-ethylenediaminetetraacetate treatment and osmotic shock, two methods known to release periplasmic enzymes.     

Polysomes in various phases of the cell cycle in synchronized plasmacytoma cells

The fraction of membrane-bound and free polysomes during different phases of the cell cycle was determined in suspension cultures of mouse plasmacytoma cells, synchronized by growth in isoleucine-deficient medium. The membrane-bound polysomes reached a maximum value (about 28 % of total polysomes) during the G 1 phase. In the S phase and G 2 phase only 18 to 20 % of the total polysomes were found to be membrane-bound. A high percentage of membrane-bound polysomes in the G 1 phase of the cell cycle agrees with the earlier finding that maximum synthesis of immunoglobulin light chain takes place on polysomes bound to the membrane in the G 1 phase of the cell cycle. The presence of a significant fraction of membrane-bound polysomes in the S and G 2 phases of the cell cycle would suggest that membrane-bound polysomes are also involved in the synthesis of proteins other than immunoglobulins.The ultrastructure of the cells during the various phases of the cell cycle was also studied. During the G 1 phase the surface of the majority of cells was distinguished by the presence of ruffles and slender villus-like cytoplasmic projections. In the S phase the surface contour tended to become smooth and even. These differences in the surface morphology may reflect the change in function which occurs during the transition from the G 1 to the S phase.     

Volume growth, murein synthesis, and murein cross-linkage during the division cycle of Escherichia coli PA3092.

Cells of Escherichia coli PA3092 were synchronized by centrifugal elutriation. The synchronously growing cells were double labeled with -3H or DL-[meso-2,6-14C]diaminopimelic acid (DAP) at different times. Cells incorporated [3H]DAP at a continuously increasing rate during their cycle, with a maximum occurring at about 30 min before division for trichloroacetic acid-precipitated cells (whole cells) and about 10 min before division for sodium dodecyl sulfate-treated cells (sacculi). This was in good agreement with the observed kinetics of volume growth under these conditions. Furazlocillin, which preferentially interacts with penicillin-binding protein 3, modified the pattern of incorporation of [3H]DAP. Electron microscopy indicated that furazlocillin did not inhibit the initiation of division but rather its completion. In addition, we measured the cross-linking of the murein inserted at different times during synchronous growth. The highest percentages were found to occur around division. At this same time, the cross-linking of old peptidoglycan was found to be decreased.     

Activity of lysosomal enzymes in the various cell cycle phases of synchronized HeLa cells.

The activities of 5 lysosomal enzymes (acid DNase, β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase and cathepsin D) were measured in HeLa cells in various cell cycle phases. The cells were synchronized either by shake-off of mitotic cells followed by resuspension in fresh medium, or by addition of amethopterin and adenosine for 16 h and reversal with thymidine. Metaphase arrest was obtained with colcemid in cells previously synchronized by means of amethopterin/thymidine. The specific activities (activity/mg protein) of the different enzymes were found to be constant following synchronization both with the shake-off technique and with the amethopterin/thymidine treatment. Furthermore, the specific enzyme activities were unaltered by metaphase arrest by colcemid. Our data indicate that lysosomal enzyme synthesis is continuous during the cell cycle of HeLa cells. The specific activity of β-glucuronidase was found to be about 3 times higher in HeLa cells grown in suspension cultures than in cells grown on solid surface. The activities of the other enzymes measured were approximately equal in suspension cells and surface cells.