Synchronous cell differentiation in Caulobacter crescentus.

The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili.     

Morphological changes in the central vacuole ofBlastocystis hominis during in vitro culture

Summary Morphological changes in the central vacuole during the growth in in vitro culture ofBlastocystis hominis were investigated by light and electron microscopy. Most cells in log phase and an early stationary phase showed a positive staining reaction in the central vacuole with PAS or Sudan black B stain, whereas cells in late stationary phase showed few positive reactions. Electron microscopic observations revealed that 95% ofB. hominis cells in log phase and 50% of cells in early stationary phase, had a substantial accumulation of electron-dense material in the central vacuole. In contrast, only 25% of the organisms in late stationary phase had an electron-dense central vacuole, while more than 50% of cells had an electron-lucent central vacuole. These results indicate thatB. hominis accumulated carbohydrates and lipids in the central vacuole during cell growth and that the organism probably consumed these metabolic substances during stationary growth. Therefore, it is strongly suggested that the central vacuole is an important organelle for storage of metabolic substances, such as carbohydrates and lipids, required for cell growth.Abbreviations PBS phosphate-buffered saline - PAS periodic acid-Schiff     

Localization of Surface Structures During Procaryotic Differentiation: Role of Cell Division in Caulobacter crescentus

Asymmetric cell division in Caulobacter crescentus produces two cell types, a stalked cell and a new swarmer cell, with characteristics surface structures. We have examined the role of the cell cycle in the differentiation of these two cells using the adsorption of bacteriophage øLC72, the assembly of the polar flagellum, and stalk formation as assays for changes in surface morphology. Previous studies of this aquatic bacterium [17, 25] have suggested that the replicating chromosome acts as a 'clock' in timing the formation of the flagellar filament at one pole of the new swarmer cell. The analysis of conditional cell cycle mutants presented here extends these results by showing that DNA synthesis is also required for adsorption of phage øLC72 and, more importantly, they also suggest that a late cell division step is involved in determining the spatial pattern in which the phage receptors and flagella are assembled. We propose that this cell division step is required for formation of 'organizational' centers which direct the assembly of surface structures at the new cell poles, and for the polarity reversal in assembly that accompanies swarmer cell to stalked cell development.     

The Extracellular Protein of Luteococcus japonicus subsp. casei Reactivates Cells Inactivated by UV Irradiation or Heat Shock

The culture liquid of Luteococcus japonicus subsp. casei was found to be able to reactivate cells of this bacterium inactivated by UV irradiation or heat shock. The antistress activity of the culture liquid was due to the presence of an extracellular exometabolite of a protein nature with a molecular mass of more than 10 kDa. When the bacterium was grown in a nutrient broth or glucose-containing mineral medium, the antistress protein was secreted by cells in the logarithmic growth phase. The reactivating effect of the antistress protein was inversely proportional to the survival rate of stressed cells.     

Chromosome replication in Caulobacter crescentus growing in a nutrient broth.

The pattern of chromosome replication in the Caulobacter crescentus cell cycle was studied by examining the rate of deoxyribonucleic acid (DNA) synthesis during synchronous growth in a fast-growth nutrient broth. As reported previously for the cell cycle in a slow-growth minimal medium (Degnen and Newton, 1972), the Caulobacter cell cycle (at the fastest available growth rate) in nutrient broth consisted of three distinct periods in terms of DNA synthetic activity. The swarmer-cell cycle consisted of a presynthetic period (G1), synthetic period (S), and postsynthetic period (G2) of 30, 50, and 35 min, respectively, whereas the stalked-cell cycle consisted of S and G2 periods of 50 and 35 min, respectively. Synchronously growing cells in the nutrient broth were stained to visualize nuclear bodies. Two nuclear bodies could be discerned in both swarmer and stalked cells, and four could be discerned in predivisional cells. DNA content per cell was determined chemically and found to be about the same in swarmer and stalked cells; it was equivalent to roughly twice the value expected from the kinetic complexity reported previously (Wood et al., 1976) for Caulobacter DNA.     

Stalkless mutants of Caulobacter crescentus.

A stalk, a single falgellum, several pili, and deoxyribonucleic acid (DNA) phage receptors are polar surface structures expressed at a defined time in the Caulobacter crescentus cell cycle. When mutants were isolated as DNA phage phiCbK-resistant or ribonucleic acid (RNA) phage phiCp2-resistant, as well as nonmotile, strains, 5 out of 30 such mutant isolates were found not to possess stalks, but did possess inactive flagella. These stalkless mutants were resistant simultaneously to both DNA and RNA phages and did not possess pili and DNA pendent stalkless mutants. All motile revertants simultaneously regained the capacity to form stalks and susceptibility to DNA and RNA phages. It is suggested that a single mutation pleiotropically affects stalk formation, flagella motility, and coordinate polar morphogenesis of pili and DNA phage receptors. The stalkless mutants grew at a generation time similar to that of the wild-type strain at 30 degrees C. Cell size and morphology of a stalkless mutant, C. crescentus CB13 pdr-819, were also similar to those of the wild-type strain, except for the absence of a stalk. In addition, the CB13 pdr-819 predivisional cells were partitioned into smaller and larger portions, indicating asymmetrical cell division, as in the wild-type strain. From these results, it is suggested that swarmer cells undergo transition to cells of a stalked-cell nature without stalk formation and that the cell cycle of the stalkless mutant proceeds in an ordered sequence similar to that defining the wild-type cell cycle.     

Effect of Rifampicin and Chloramphenicol on Progeny Single-stranded DNA Synthesis of Bacteriophage ϕX174

Neither bacteriophage ?X174 single-stranded DNA synthesis nor phage growth was affected by rifampicin (200 μg/ml) once it started, whereas a low concentration of chloramphenicol (30 μg/ml) inhibited the phage growth when added in a late phase of infection. When rifampicin was added at a stage where double-stranded duplex (RF) DNA replication proceeded preferentially in the presence of chloramphenicol, or even after chloramphenicol was removed before the addition of rifampicin, both single-stranded DNA synthesis and phage growth were inhibited. These results suggest that RNA synthesis sensitive to rifampicin was necessary to initiate single-stranded DNA synthesis, but no longer needed once ?X174 DNA synthesis started.     

Cell Size,Macromolecule Composition,Nuclear Number,Oxygen Consumption and Cyst Formation During Two Growth Phases in Unagitated Cultures of Acanthamoeba castellanii1

SYNOPSIS. The size, composition and physiology of average cells have been studied in cultures of Acanthamoeba castellanii during the phases of logarithmic growth and population growth deceleration (PGD). Most of the features examined were relatively constant during log phase, but had significant changes during PGD. Average cell volume increased about 60% and total dry mass about 15–20% during the latter period. Total protein content remained constant thruout both growth phases, but cytochrome oxidase doubled during PGD. DNA, RNA and glycogen levels began to change during late log phase. DNA decreased about 50% and RNA increased about 75%. Glycogen decreased 50% during the RNA build-up and then increased to a plateau above the log phase level. A final decrease in glycogen followed an increase in the relative numbers of cysts in late PGD. It was found that PGD begins when O₂ becomes limiting and evidence that the subsequent changes in macromolecule composition are related to encystation is discussed.     

Transfer and expression of pseudomonas plasmid RP1 in Caulobacter.

This study demonstrates that the host range of Pseudomonas plasmid RP1 includes the genus Caulobacter. Caulobacter was shown to acquire three antibiotic resistance markers located in RP1. A fourth plasmid marker, susceptibility to an RNA bacteriophage, was not expressed, but could be transferred from Caulobacter to Escherichia coli. The lack of phenotypic expression of the phage marker was manifested by the inability of the phage to adsorb or to produce plaques on Caulobacter transcipients. Matings of Pseudomonas aeruginosa and Caulobacter vibrioides CV6 were carried out in the presence of bacteriophage phi6, a DNA phage that infects and kills only swarmer cells of Caulobacter. No decrease in plasmid transfer in the presence of phage phi6 was detected, suggesting that stalked cells, and not swarmer cells, serve as recipients. Our evidence suggests that transfer of chromosomal segments from Caulobacter may be mediated by plasmid RP1; such segments are not stably maintained.     

Ribosome biosynthesis in Tetrahymena thermophila. III. Regulation of ribosomal RNA degradation in growing and growth arrested cells

We have measured the turnover rate of ribosomal RNA in exponentially growing Tetrahymena thermophila cells, cells entering the plateau phase of growth, and nutrient-deprived (starved) cells. Ribosomal RNA is stable in cells in early log phase growth but it begins to turnover as the cells begin a deceleratory growth phase prior to entering a plateau state. Likewise, rRNA in cells transferred from early log phase growth to a starvation medium begins to be degraded immediately upon starvation. In both cases the degradation of rRNA exhibits biphasic kinetics. A rapid initial exponential degradation with a half time of nine and one-half hours lasting for six hours is followed by a slower exponential degradation with a half-life of 35 hours. When starved cells are transferred to fresh growth medium turnover of rRNA ceases. The evidence presented suggests that the alteration in degradation rate is a regulated process which is most likely independent of the cell cycle.     

Phage formation in Staphylococcus muscae cultures; nucleic acid synthesis during virus formation

1. The total nucleic acid synthesized by normal and by infected S. muscae suspensions is approximately the same. This is true for either lag phase cells or log phase cells. 2. The amount of nucleic acid synthesized per cell in normal cultures increases during the lag period and remains fairly constant during log growth. 3. The amount of nucleic acid synthesized per cell by infected cells increases during the whole course of the infection. 4. Infected cells synthesize less RNA and more DNA than normal cells. The ratio of RNA/DNA is larger in lag phase cells than in log phase cells. 5. Normal cells release neither ribonucleic acid nor desoxyribonucleic acid into the medium. 6. Infected cells release both ribonucleic acid and desoxyribonucleic acid into the medium. The time and extent of release depend upon the physiological state of the cells. 7. Infected lag phase cells may or may not show an increased RNA content. They release RNA, but not DNA, into the medium well before observable cellular lysis and before any virus is liberated. At virus liberation, the cell RNA content falls to a value below that initially present, while DNA, which increased during infection falls to approximately the original value. 8. Infected log cells show a continuous loss of cell RNA and a loss of DNA a short time after infection. At the time of virus liberation the cell RNA value is well below that initially present and the cells begin to lyse.     

The extracellular protein of Luteococcus japonicus subsp casei reactivates cells inactivated by ultraviolet radiation or heating

The culture liquid of Luteococcus japonicus subsp. casei was found to be able to reactivate cells of this bacterium inactivated by UV irradiation or heat shock. The antistress activity of the culture liquid was due to the presence of an extracellular exometabolite of a protein nature with a molecular mass of more than 10 kDa. When the bacterium was grown in nutrient broth or glucose-containing mineral medium, the antistress protein was secreted by cells in the logarithmic growth phase. The reactivating effect of the antistress protein was inversely proportional to the survival rate of stressed cells.     

Inhibition of bacterial cell wall mucopeptide synthesis: A new function of RNA bacteriophage Qβ

Studies of the morphology of Escherichia coli showed that these bacilli when infected with RNA phage Qβ in broth containing hypertonic sucrose and Mg²⁺ formed osmotically labile spherical cells or spheroplasts. Phage-induced spheroplasts readily released their burst of phage when diluted into ordinary culture broth without sucrose. Investigation of the mechanism of host cell lysis revealed that incorporation of [³H]diaminopimelic acid (DAP) into the mucopeptide layer of the cell wall was markedly inhibited starting at about the midpoint of the phage replication cycle. The major site of inhibition is the DAP-containing mucopeptide layer since the synthesis of the lipoprotein-lipopolysaccharide layer, making up the bulk of the cell wall of E. coli, was not affected. A model for Qβ-mediated cell lysis is discussed which is analogous to penicillin-induced cell rupture.     

Unidirectional polar growth of cells of Seliberia stellata and aquatic seliberia-like bacteria revealed by immunoferritin labeling

When grown in a complex peptone-yeast extract culture medium, Seliberia stellata and related morphologically similar aquatic bacterial strains typically divided asymmetrically, giving rise to a motile swarmer and a longer sessile rod. Indirect immunoferritin labeling of these bacteria, followed by incubation during which cell growth occurred, has provided evidence that antigenic cell-surface components are synthesized de novo in a sharply demarcated zone at one pole of the growing parent cells. Cell elongation occurred unidirectionally from the pole showing the de novo surface synthesis; it was this end of the elongating, helically sculptured (i.e., screw-like) rod that became the daughter swarmer cell. The daughter swarmers, produced after polar growth and division of the immunoferritinlabeled parent cells, were not labeled. The immunoferritin label remaining on the parent cell did not appear to be diluted or disturbed by the cell growth and division process. Under the cultural conditions used in this study, the growth and division events which led to production of swarmer cells in the seliberia strains examined met two major criteria of accepted definitions of budding (de novo cell surface synthesis and transverse asymmetry of division). However, the developing daughter cell was not initially narrower than the parent and thus did not increase in cell diameter during growth.In memory: R. Y. Stanier     

The Fate of RNA Degradation Products in Starved Cultures of Tetrahymena pyriformis W.*

The RNA content of a population of Tetrahymena pyriformis W was followed during the growth phases of the culture. The cellular RNA levels were found to reach a maximum in early log phase and to decrease throughout the remainder of the log and deceleration phases. There was a 25% decrease in RNA amount when cells in late stationary phase were compared to those in deceleration. This loss of RNA was mimicked when cells from the deceleration phase were suspended in a non-nutrient buffered medium. Procedures were established to determine RNA content and the intra- and extracellular distribution of RNA degradation products, namely purine and pyrimidine bases and orthophosphate. Balance sheets are presented to show that the decrease in RNA levels was accompanied by an equivalent increase in purine and pyrimidine bases and phosphorus derivatives. The validity of the procedures employed was demonstrated. The influence of magnesium, cholesterol and glucose on the cells suspended in a non-nutrient buffer was examined. Each was found to affect the ultimate distribution of RNA products in a characteristic fashion suggesting that each compound acts by a different mode of action.     

Lipase production of Staphylococcus carnosus in a dialysis fermentor

Summary In a newly constructed one-vessel dialysis fermentor, a strain of Staphylococcus carnosus TM300 carrying the lipase secretion plasmid pLipPS1 was used to investigate exoenzyme and biomass production. The bacterial culture grows in an inner compartment of 21 volume, separated from a 101 nutrient broth compartment by a conventional dialysis membrane. In order to avoid substrate depletion and to prolong the growth phase, a highly concentrated nutrient broth was used. The biomass production reached 60 g cell dry weight/l. The increase in extracellular lipase concentration was directly coupled with the increase of cell mass and reached a value of 230 mg/l culture supernatant. Harvesting the cells in the late growth phase, the lipase content was about 30% of the total exoproteins in the supernatant.     

Factors Affecting Transfection in Bacillus stearothermophilus

The conditions for the infection of Bacillus stearothermophilus 4S with TP-1C phage deoxyribonucleic acid (DNA) are described. Cells from log-phase cultures are the most susceptible to phage DNA infection (transfection). A cellular component (competence factor) which enhances transfection is released into the culture medium during the transition period between the log and stationary phase of growth. Transfection is stimulated in the order of decreasing effectiveness, by Fe(3+), Mn(2+), and Mg(2+). The efficiency of transfection is the highest in cells growing at 60.5 C and does not occur in cells growing at 67 C although the cells are growing normally. A cellular component (competence factor) of this organism, which is released into the culture medium, advances by 40 min some step in the uptake of phage DNA.     

Bacterial growth and the concentrations of cyclic nucleotides inLegionella pneumophila cultures

The growth kinetics ofLegionella pneumophila, and the rates of DNA and RNA synthesis in this organism, are qualitatively similar to that of other gram negative organisms of clinical importance. After a brief stationary phase, culture viability is rapidly lost. This loss of viability is not associated with detectable cell lysis or with significant breakdown of cellular DNA; it is, however, preceded by the rapid breakdown of approximately 50% of the cumulatively labeled RNA. During logarithmic growth in buffered yeast extract broth, bacterial cells secreted into the medium large amounts of cyclic GMP at both 37°C and room temperature, while those grown in a buffered synthetic broth did not. Cyclic AMP increased moderately during stationary phase at 37°C and room temperature in both media. Neither added cyclic AMP nor added cyclic GMP influenced the rate of growth ofL. pneumophila in either broth, with or without 1-methyl-3-isobutyl-xanthine, a phosphodiestease inhibitor. Dibutyrul cyclic AMP and 8-bromo cyclic GMP individually or in combination also had no effect on growth.     

Cell-cycle-dependent polar morphogenesis in Caulobacter crescentus: roles of phospholipid, DNA, and protein syntheses.

During swarmer cell differentiation in Caulobacter crescentus, morphogenesis at the swarmer pole is characterized by the loss of the flagellum, by the loss of phage receptor activity (PRA) (the ability of the cell to adsorb phage phi CbK), and finally by the initiation of stalk outgrowth at the site formerly occupied by the flagellum and the PRA. We show here that each of these events is a cell cycle-dependent event requiring continuous protein synthesis for its execution but occurring normally in the absence of DNA synthesis or phospholipid synthesis. During stalked-cell differentiation, the flagellum and PRA reappear and the stalk elongates considerably. We show here that these events are also cell cycle dependent, requiring not only de novo protein synthesis but also DNA and phospholipid syntheses. When synchronous cells dividing 160 min after collection were used, PRA reappearance occurred at 110 min. This PRA reappearance was dependent on a phospholipid synthesis-requiring event occurring at 70 min, a DNA synthesis-requiring event occurring at 95 min, and a protein synthesis-requiring event occurring at 108 min. In the absence of net phospholipid synthesis, stalk elongation appeared more or less normal, but the stalks eventually became fragile, and by 240 min, most of the stalks had broken off, leaving only stubs attached to the cell body.     

Synthesis of photoreactivating enzyme in synchronous and asynchronous cultures of Escherichia coli

The technique of flash photolysis was used to study cellular variations in the number of photoreactivating enzyme (PRE) molecules during the cell division cycle of the UV-sensitive E. coli strain B_S?1. No variations in the number of PRE molecules per genome were observed throughout the cell division cycle when synchronized cells cultured in either glucose-minimal or succinate-minimal medium were used. This is interpreted to mean that PRE synthesis is continuous throughout the cell cycle for glucose-grown cells, but may stop at the time chromosome replication ceases prior to division, in succinate-grown cells. The effect of growth rate and stage of growth on cellular PRE content in asynchronous cultures was also determined. Variations in the number of PRE per genome were observed for both synchronous and asynchronous cells cultured in different media and occurred in a manner that suggested a dependence on growth rate. PRE per genome increased with generation time. Stationary phase cells from each culture medium (nutrient broth, glucose-minimal, succinate-minimal) had more PRE per genome than did respective log phase cells. It is suggested that PRE synthesis may be controlled by some aspect of chromosome replication.