Macromolecular synthesis and cell division during morphogenesis of Arthrobacter crystallopoietes

The sphere-rod-sphere morphology cycle of Arthrobacter crystallopoietes was accompanied by changes in the rate of growth and the rates of DNA, RNA and protein synthesis. The patterns of macromolecule synthesis resembled those found in other bacteria during a step-up followed by a step-down in growth rate. During the step-up in growth spherical cells grew into rods and macromolecules were synthesized in the absence of cell division. During stepdown, successive rounds of septation produced progressively smaller cells which did not separate and remained in chains. The morphology of the cells was dependent on the growth rate and could be altered by changing the dilution rate in a malate-limited chemostat. Gradual transitions in morphology and gradual increases in macromolecule content of the cells occurred as the growth rate was increased in the chemostat. Sphere to rod morphogenesis occurred when DNA synthesis was inhibited by treatment with mitomycin C or by thymine starvation. The DNA-deficient rods did not divide and eventually lysed. DNA, RNA and protein synthesis were continuously required for the reductive division of rods to spheres.Abbreviations MS mineral salts - GS mineral salts plus glucose - CA casamino acids - GSCA mineral salts plus glucose plus casamino acids - cAMP cyclic adenosine-3,5-monophosphate - RNA ribonucleic acid - DNA deoxyribonucleic acid     

Sphere-rod morphogenesis in Arthrobacter crystallopoietes. II. Peptides of the cell wall peptidoglycan

Cell walls of Arthrobacter crystallopoietes grown as spheres and as rods were solubilized by treatment with the B enzyme from Chalaropsis, an N-acetylmuramidase. The neutral glycopeptides were then isolated by chromatography on ECTEOLA cellulose. The glycopeptides, consisting of disaccharide-peptide units interlinked by peptide cross-bridges, were fractionated by gel filtration on Sephadex columns into oligomers of various sizes. The size distribution ranged from monomers with no cross-bridges to polymers with a high degree of polymerization, but did not differ significantly between cell walls from cells grown as spheres or rods. Some small differences in the distribution of C- and N-terminal amino acids were found. Analyses revealed that all the peptide bridges in the glycopeptide fractions from rod cell walls were formed by one l-alanine residue. In sphere cell walls, l-alanine was also found, but, in addition, higher oligomers of the glycopeptide contained glycine in their cross-bridges. These results were confirmed by determinations of C- and N-terminal amino acids released after lysostaphin and AL-1 enzyme digestions and by Edman degradations. Models representing the structures of the sphere and rod cell walls are presented. These structures indicate that the sphere cell wall is probably a more loosely knit macromolecule than is the rod cell wall.     

Stability of enzymes in starving Arthrobacter crystallopoietes.

Cell-free extracts prepared from spherical and rod-shaped cells of Arthrobacter crystallopoietes were assayed for enzymes during various periods of starvation. The level of NADH oxidase dropped to 20 and 30%, respectively, in spherical and rod-shaped cells during the first 1 to 2 days of starvation and then remained constant for 9 days. Catalase activity decreased continuously and reached a low level in 9 days. Enzymes involved in glucose metabolism and the tricarboxylic acid cycle were stable for the duration of the experiment (about 1 week). Succinic dehydrogenase, fumarase and aconitase were stable during 21 days of starvation, which is the longest time enzymes have been shown to be stable in any bacterium under conditions of total starvation.     

Alteration of glucose metabolism of Arthrobacter crystallopoietes by compounds which induce sphere to rod morphogenesis

Succinate and several other compounds which induce sphere to rod morphogenesis of A. crystallopoietes were found to suppress both catabolism and assimilation of glucose. Diauxic growth patterns resulted from growth on glucose plus any one of these compounds. Glutamate stimulated growth but was not an inducer of morphogenesis. With this compound, diauxic growth and suppression of glucose catabolism or assimilation did not occur. Glucose permease was studied with alpha-methylglucoside as substrate. The entry system for glucose was found to involve active transport and to have a K(m) of 8 x 10(-4)m. It was inducible, was repressed in succinate-grown cells, and was also inhibited by succinate. The exit system was constitutive and appeared to be less sensitive than the entry system to inhibition by azide. The properties of the glucose permease system may account for the slow growth of the organism on glucose and the preferred use of other substrates for growth. Studies of metabolic pathways for glucose metabolism indicated the operation of the Embden-Meyerhof-Parnas (EMP) and pentose phosphate pathways and of the tricarboxylic acid cycle. Cells grown on glucose plus limiting amounts of succinate or other inducers of morphogenesis metabolized the glucose only after exhaustion of the inducers. Under these circumstances, the organisms employed the EMP pathway to a greater extent than when growing on glucose as sole carbon source.     

Adenylate nucleotide levels and energy charge in Arthrobacter crystallopoietes during growth and starvation

The adenylate nucleotide concentrations, based on internal water space, were determined in cells of Arthrobacter crystallopoietes during growth and starvation and the energy charge of the cells was calculated. The energy charge of spherical cells rose during the first 10 h of growth, then remained nearly constant for as long as 20 h into the stationary phase. The energy charge of rod-shaped cells rose during the first 4 h of growth, then remained constant during subsequent growth and decreased in the stationary growth phase. Both spherical and rod-shaped cells excreted adenosine monophosphate but not adenosine triphosphate or adenosine diphosphate during starvation. The intracellular energy charge of spherical cells declined during the initial 10 h and then remained constant for 1 week of starvation at a value of 0.78. The intracellular energy charge of rod-shaped cells declined during the first 24 h of starvation, remained constant for the next 80 h, then decreased to a value of 0.73 after a total of 168 h starvation. Both cell forms remained more than 90% viable during this time. Addition of a carbon and energy source to starving cells resulted in an increase in the ATP concentration and as a result the energy charge increased to the same levels as found during growth.Nonstandard Abbreviations cGMP 3,5 guanosine monophosphate - ppGpp guanosine tetraphosphate - MS mineral salts - HC casein hydrolysate - TEA triethanolamine buffer - Pi inorganic phosphate     

Electron microscopic observations of cell division in Mycobacterium vaccae V1

Cell division of Mycobacterium vaccae was initiated by deposition of new wall material in the cross wall. The surface layers of the old wall remained continuous until septum formation was complete. Subsequently, rupture of the outer cell wall layers occurred circumferentially, leaving rings on the cell wall. The two daughter cells remained connected with each other at the new pole and bent to form V-shaped structures at the connecting point.     

Isolation and characterization of morphogenetic mutants of Arthrobacter crystallopoietes

Mutants of Arthrobacter crystallopoietes that exhibited altered ability to undergo the normal sphere-to-rod-to-sphere morphogenetic cycle were isolated. The procedure used to isolate these mutants involved velocity sedimentation in a sterile sucrose gradient to separate morphogenesis-deficient spherical cells from rod-shaped cells capable of normal morphogenesis. Three classes of mutants were obtained: (i) those that cannot form rods, (ii) those that cannot form long rods, and (iii) those that form long rods but exhibit more extensive rudimentary branching than the wild type. The isolation and characterization of these mutants are described, and the use of these mutants in the study of the morphogenetic cycle of arthrobacters is discussed.     

Adenosine triphosphate pool levels and endogenous metabolism in Arthrobacter crystallopoietes during growth and starvation

The adenosine triphosphate (ATP) content of Arthrobactery crystallopoietes was measured during growth, starvation and recovery from starvation. During exponential growth of the cells as spheres in a glucose salts medium, the level of ATP per cell remained constant at 8.0×10^-10 g/cell. Morphogenesis to rodshaped cells and an increased growth rate following addition of casein hydrolysate was accompanied by an almost two-fold increase in the ATP level. As division of the rod-shaped cells proceeded, the level of ATP declined. After growing as rods for 12–14 h the cells underwent fragmentation to spheres during which time the ATP level again increased to the original value of 8.0×10^-10 g/cell. As the spherical cells resumed growth on the residual glucose, their ATP content declined for a short period and then remained relatively constant. During starvation of sphere or rod-shaped cells for one week, the ATP level declined by approximately 70% during the first 40–50 h and then remained constant. The endogenous metabolism rate of spherical cells declined during the first 10–20 h of starvation and then remained constant at approximately 0.02% of the cell carbon being utilized per h. Addition of glucose to spherical cells which had been starved for one week increased both the ATP content per cell and their rate of endogenous metabolism. The ATP content fluctuated and then remained at a level higher than maintained during starvation while endogenous metabolism quickly declined.Non-Standard Abbreviations ATP adenosine triphosphate - GS glucose mineral salts - HC casein hydrolysate - PVP polyvinylpyrrolidone - DMSO dimethylsulfoxide - MOPS morpholinopropane sulfonic acid - EDTA ethylene diaminetetraacetic acid     

Electron microscopic study of plant-animal cell fusion

Summary Avian erythrocytes and protoplasts isolated from mesophyll cells of tobacco plants were suspended in 1% protease, agglutinated with polyethylene glycol (PEG) and subsequently fused upon elution of the PEG. The fusion reaction was monitored by scanning (SEM) and transmission (TEM) electron microscopy. SEM studies showed a marked difference in the topography of agglutinated cells. During, and subsequent to fusion, the markedly different surfaces of the two cell types became homogeneous and lines of demarcation between the cells were no longer visible. TEM revealed that adhesion occurred over the entire membrane area between agglutinated cells. Incipient fusion was evidenced by the appearance of vacuoles at the intermembrane surfaces. During initial elution of the PEG, cytoplasmic channels between erythrocytes and protoplasts were evident. With continued elution of the PEG, starch-containing plant chloroplasts and starch grains were seen within erythrocytes and homogenous erythrocyte cytoplasm was present inside plant protoplasts. Cytoplasmic mixing between the two cell types occurred within 3 hours of elution. The frequency of interkingdom fusion was estimated to be 0.5–1%.     

Degradation of pyridine by Arthrobacter crystallopoietes and Rhodococcus opocus strains

Abstract Two pyridine-degrading microorganisms Arthrobacter crystallopoietes (VKM Ac-1334D) and Rhodococcus opacus (VKM Ac-1333D) were isolated from soil. The Gas chromatography-mass spectroscopy analysis showed that the former species formed 3-hydroxypyridine, 2,3- and 2,6-dihydroxypyridines during its growth in media containing pyridine, while the latter formed 2-hydroxy- and 2,6-dihydroxypyridines as degradation intermediates. Products of the pyridine ring cleavage (5-amino-2-oxo-4-pentenoic acid and 3-pentenoic acid monoamide) were also detected.     

Lipid Composition of Growing and Starving Cells of Arthrobacter crystallopoietes

The lipid composition of growing and starving cells of Arthrobacter crystallopoietes was compared. Although the lipid composition of the two cell types was similar, the amount of total lipids recovered from the starving cells was 30.4% less than that recovered from the growing cells. The loss of lipids, as compared to the loss of total cell mass during starvation, was (i) proportional to the loss of the cell mass (phosphatidylinositol, phosphatidylglycerol-2, and cardiolipin), (ii) greater than the loss in cell mass (neutral lipids, "glycophospholipids," and phosphatidic acid), or (iii) less than the loss in cell mass (coenzyme Q, glycolipids, and phosphatidylglycerol-1).     

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant.

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.     

Electron microscopic study of alpha-actinin.

Electron microscopic studies of the structure of purified α-actinin alone and in complex with F-actin have determined the molecular shape and size of this protein. α-Actinin molecules represent rods of about 300 Å in length and about 20 Å in diameter.     

Long-Term Starvation Survival of Rod and Spherical Cells of Arthrobacter crystallopoietes

Spherical and rod-shaped cells of Arthrobacter crystallopoietes, harvested during exponential growth, were subjected to total starvation for periods of time as long as 80 days. Viability measurements were made by plate count and slide culture procedures. Both cell forms remained 100% viable for 30 days. Thereafter, viability of rods and spheres decreased equally at a slow rate. After 60 days of starvation, more than 65% of both cell forms were viable. No significant cell lysis occurred as evidenced by microscopic examination, the small amount of 260-nm absorbing material found in the starvation buffer, and stability of radioactively labeled deoxyribonucleic acid in the cells. Endogenous respiration decreased 80-fold during the first 2 days of starvation, accompanied by a 30% decrease in dry weight of the cells. Thereafter, cellular carbon was oxidized to CO(2) at the constant level of 0.03%/hr over the remaining 78-day starvation period.     

Electron microscopic study of the process of intermediate cell division in the cyst of the coccidian Sarcocystis muris

The intermediate cell is a third definitely outlined morpho-functional type of cells within sarcocysts, in addition to the two other well known ones--metrocytes and merozoites (Fedoseenko, Levit, 1979; Beyer et al., 1981). The intermediate cell divides by endodyogeny, the nuclear division being accomplished by semi-closed pleuromitosis. In the dividing nuclei, centrioles and extranuclear bundle of microtubules connecting two pairs of centrioles are seen in addition to centrocones and associated semi-spindles. Pro-, ana- and telophases of mitosis have been followed. The microtubule organizing center (MTOC) seen in the cytoplasm of the intermediate cell is represented by the polar ring with microtubules originating from it. The MTOC is involved in the division of both the nucleus and the cytoplasm. The formation of the polar ring (MTOC) from the Golgi-adjunct has been first discovered and followed in the course of the intermediate cell division.     

A scanning electron microscopic and x-ray microanalytic study of cell surface material during amphibian neurulation.

Treatment with lanthanum (La3+) after fixation in phosphate (PO4-3)-buffered glutaraldehyde results in the deposition of a cell surface material (CSM) primarily on the developing urodele amphibian neural axis. X-ray probe microanalysis indicates that calcium (CA2+) levels are considerably higher in the neural fold region. La3+ displaces Ca2+ from negatively-charged moieties on biological membranes. Once bound, La3+ likely interacts with residual phosphate(s) resulting in deposition of CSM. Elemental X-ray microanalysis shows CSM contains mostly lanthanum and phosphorus. The high level of regional La3+ binding is correlated with inherently greater Ca2+ levels in the developing neural axis.