Superoxide dismutase in vegetative cells, heterocysts and akinetes of Anabaena cylindrica Lemm

Abstract: Superoxide dismutase (SOD) activity was assayed in vegetative cells, heterocysts and akinetes of Anabaena cylindrica Lemm. The iron-containing isoenzyme (Fe-SOD) was in all cases predominant over the manganese-containing isoenzyme (Mn-SOD). Differentiated cells maintained the same relative content of the two enzymes as in vegetative cells. However, heterocysts and akinetes contained only 20 and 35%, respectively, of the total SOD activity present in vegetative cells.
Both Mn-SOD and Fe-SOD activities increased in all types of cells isolated from A. cylindrica grown at high light intensity. The increase of SOD in heterocysts paralleled that of nitrogenase, suggesting a role of SOD in the protection mechanism of nitrogenase.     

Glycolaldehyde Dehydrogenase,Its Involvement in Vitamin B6 Biosynthetic Pathway of Escherichia coli B

The deoxyribonucleic acid (DNA) polymerases were partially purified from spores and vegetative cells of Bacillus subtilis. Some biochemical properties of the enzymes from the spores were studied in comparison with those from the vegetative cells. The spores and vegetative cells had at least three species of DNA polymerases (DNA polymerase I, II and III). These DNA polymerases in spores could not be distinguished from those in vegetative cells, respectively, with regard to the reresponses to ionic strength, the sensitivity to thiol-blocking agents, the template specificity, pH and temperature optima in assay, and the sedimentation behavior. It is inferred that DNA polymerases from spores was essentially identical to those from vegetative cells.

The DNA polymerase activity decreased rapidly in the course of sporulation, and only about 20% is recovered in the spores, suggesting that an extentive inactivation mechanism of the enzymes would be involved during sporulation.     

Protein kinases of Dictyostelium discoideum, strain AX-2.

In cell homogenates of Dictyostelium discoideum, strain AX-2, four major soluble protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) and one membrane-associated protein kinase activity were identified. The enzymes showed high affinity for casein. One of the enzymes was purified by affinity chromatography on casein-coated Sepharose. The soluble high molecular weight enzymes phosphorylated histones, whereas the low molecular weight enzymes did not. The same protein kinase species were present in vegetative and aggregation-competent cells. Their specific activity, however, changed during the development to aggregation competence. None of the enzymes was stimulated by cyclic AMP or cyclic GMP, regardless of their origin from vegetative or aggregation-competent cells.     

Two polypeptides associated with the ribonucleic acid polymerase core of Bacillus subtilis during sporulation.

The ribonucleic acid (RNA) polymerase from log-phase and sporulating cells of Bacillus subtilis was analyzed to determine whether any structural changes occurred during sporulation. The elution pattern of RNA polymerase from a deoxyribonucleic acid (DNA)-cellulose column revealed that sporulating cells at stages III and IV contained a new RNA polymerase fraction in addition to the vegetative holoenzyme (alpha2betabeta'sigma). Stage III cells contained the vegetative holoenzyme and a new enzyme with the composition alpha2betabeta'delta1; the molecular weight of delta1 was 28,000. Stage IV cells contained the vegetative holoenzyme, the delta1-containing enzyme, and another enzyme with the composition alpha2betabeta'delta2. The delta2 factor had a molecular weight of around 20,000. The delta-containing enzymes have a higher affinity for the DNA-cellulose column and a higher specific activity on various templates than vegetative holoenzyme. The simultaneous appearance of these enzymes with vegetative holoenzymes in sporulating cells is consistent with the data found previously with DNA-RNA hybridization studies, which showed that sporulating cells contained both vegetative and sporulation messenger RNAs.     

Isolation, Properties, Function, and Regulation of Endo-(1 → 3)-β-Glucanases in Schizosaccharomyces pombe

Cell-free extracts, membranous fractions, and cell wall preparations from Schizosaccharomyces pombe were examined for the presence of (1 → 3)-β-, (1 → 3)-α-, and (1 → 6)-β-glucanase activities. The various glucanases were assayed in cells at different growth stages. Only (1 → 3)-β-glucanase activity was found, and this was associated with the cell wall fraction. Chromatographic fractionation of the crude enzyme revealed two endo-(1 → 3)-β-glucanases, designated as glucanase I and glucanase II. Glucanase I consisted of two subunits of molecular weights 78,500 and 82,000, and glucanase II was a single polypeptide of 75,000. Although both enzymes had similar substrate specificities and similar hydrolytic action on laminarin, glucanase II had much higher hydrolytic activity on isolated cell walls of S. pombe. On the basis of differential lytic activity on cell walls, glucanase II was shown to be present in conjugating cells and highest in sporulating cells. Glucanase II appeared to be specifically involved in conjugation and sporulation since vegetative cells and nonconjugating and nonsporulating cells did not contain this enzyme. The appearance of glucanase II in conjugating cells may be due to de novo enzyme synthesis since no activation could be demonstrated by combining extracts from vegetative and conjugating cells. Increased glucanase activity occurred when walls from conjugating cells were combined with walls from sporulating cells. Studies with trypsin and proteolytic inhibitors suggest that glucanase II exists as a zymogen in conjugating cells. A temperature-sensitive mutant of S. pombe was isolated which lysed at 37°C. Glucanase activity was higher in vegetative cells held at 37°C than cells held at 25°C. Unlike the wild-type strain, this mutant contained glucanase II activity during vegetative growth and may be a regulatory mutant.     

Distribution of peptidoglycan synthetase activities between sporangia and forespores in sporulating cells of Bacillus sphaericus.

Sporulating cells of Bacillus sphaericus 9602 containing fully engulfed forespores at different stages of maturity were broken by ultrasonic disruption, followed by grinding with alumina. In this way soluble enzymes derived mainly from the sporangial or from the forespore cytoplasms were obtained. Diaminopimelate ligase activity is required exclusively for cortical peptidoglycan synthesis, is absent during vegetative growth, and is synthesized during forespore maturation. It is found exclusively in the sporangial cytoplasm. L-lysine ligase is required for vegetative cell wall peptidoglycan synthesis but not for cortex synthesis. It is found in both fractions, but it has a fourfold higher specific activity in the forespore cytoplasm. Other enzymes that are required for synthesis of the nucleotide-pentapeptide precursors of both cortical and vegetative cell wall peptidoglycans are found in similar specific activities in both compartments. Mature spores, free of any residual sporangial material, have specific activities of all of these enzymes and of L-lysine ligase similar to those in forespores and in vegetative cells and are devoid of diaminopimelate ligase activity. Thus, the differential expression of at least one gene required for spore cortex synthesis in B. sphaericus occurs exclusively in the sporangial cytoplasm.     

Variation of (1 leads to 3)-beta-glucanases in Saccharomyces cerevisiae during vegetative growth, conjugation, and sporulation.

The total (1 leads to 3)-beta-glucanase activities associated with cell extracts and cell walls of Saccharomyces cerevisiae were measured during vegetative growth, conjugation, and sporulation. Using a system of column chromatography, we resolved (1 leads to 3)-beta-glucanase activity into six different enzymes (namely, glucanases I, II, IIIA, IIIB, IV, and V). The contributions of the individual enzymes to the total activity at the different stages of the life cycle were determined. Total glucanase activity increased during exponential growth and decreased in stationary resting-phase cells. Glucanase IIIA was the predominant enzyme in stationary resting-phase cells. Glucanases I, II, IIIB, and IV were either absent or present at low levels in stationary phase cells, but their individual activities (in particular, glucanase IIIB activity) increased substantially during exponential growth. Total (1 leads to 3)-beta-glucanase activity did not change significantly during conjugation of two haploid mating strains, S. cerevisiae 2180A and 2180B, and no notable changes were detected in the activities of the individual enzymes. Sporulation was accompanied by a rapid increase and then a decrease in total glucanase activity. Most of the increase was due to a dramatic rise in the activity of glucanase V, which appeared to be a sporulation-specific enzyme. Glucanase activity was not derepressed by lowering the glucose concentration in the growth medium.     

Studies of sulfate utilization by algae

Summary Several aspects of amino acid metabolism were studied in the fruiting myxobacterium Myxococcus xanthus. Alanine and aspartate aminotransferases were detected at significant levels in vegetative cells and myxospores. In contrast, glutamate dehydrogenase, alanine dehydrogenase and aspartase were not detectable in the same preparations, which is consistent with the fact that inorganic nitrogen is not required for growth. The data presented suggest that the aminotransferases demonstrated provide for the synthesis of nonessential amino acids and concomitantly, oxidizable substrates.Isocitrate lyase activity was found in glycerol induced myxospores, but not in vegetative cells grown on two per cent Casitone medium. The emergence of isocitrate lyase in myxospores would indicate a metabolic shift toward the biosynthesis of compounds not required during vegetative growth. However, the presence of isocitrate lyase activity in vegetative cells grown in defined medium suggests that the amino acids present in the growth medium contribute to the formation of pyruvate and acetate and that glyoxylate enzymes are subject to repression when cells are grown on Casitone medium. Also, that expression of glyoxylate enzymes is not specific to myxospore formation.Based on a thesis submitted by the senior author in partial fulfillment of the requirements for the M.S. degree in Microbiology, August, 1968.     

Location of peptidoglycan lytic enzymes in Bacillus sphaericus.

The level of three peptidoglycan hydrolases was determined in the mother cell compartment and forespores of Bacillus sphaericus. Vegetative and sporulating cells contained in LD-carboxypeptidase active only on the vegetative cell wall peptidoglycan, and we have previously shown that sporulation is accompanied by the production of two new enzymes active only on the spore cortex peptidoglycan. These gamma-D-glutamyl-meso-diaminopimelate endopeptidase and a meso-diaminopimelate-D-alanine dipeptidase. The LD-carboxypeptidase activity appeared to be located in the membranes of both the mother cells and forespores. Endopeptidase activity was located in the integument fraction of the forespores, and the dipeptidase activity was only found in the forespore cytoplasm. These different locations comply with the probable different functions of these enzymes.     

Effects of glucosamine on lysis, glycerol formation, and sporulation in Myxococcus xanthus.

Glucosamine (GlcN), which has previously been shown to rescue fruiting body formation, lysis, and sporulation in a developmental mutant (G. Janssen and M. Dworkin, Dev. Biol. 112:194-202, 1985), induced lysis in vegetative and developing wild-type cells and inhibited fruiting body formation. It also resulted in a transient, intracellular increase in the concentration of glycerol, a known sporulation inducer, and sporulation of the surviving cells. Phospholipase activity, which was shown to be normally developmentally regulated, increased 7.6-fold after treatment of vegetative cells with 50 mM GlcN. Likewise, autocidal activity, which normally increased 18 to 24 h after the initiation of development, increased 20% when vegetative or developing cells were exposed to GlcN. Two mutants resistant to GlcN-induced lysis (MD1021 and MD1022) were isolated and showed neither an increase in autocide production nor an increase in phospholipase activity in response to added GlcN. MD1021 was developmentally deficient, and GlcN rescued fruiting body formation as well as phospholipase activity and autocide production. We propose that GlcN exerts its lytic effect by regulating the activity of phospholipase enzymes that release autocides, compounds that are believed to be responsible for developmental autolysis. GlcN-induced sporulation was found to depend on several factors: the initial cell density, the amount of lysis induced by GlcN, and the presence of tan-phase variants. An initial cell density of greater than 2 x 10(5) cells per ml was required to support GlcN-induced sporulation, and sporulation did not occur unless 50 to 75% of these cells had lysed. Mutants that were resistant to GlcN-induced lysis also did not sporulate in the presence of GlcN. The effects of GlcN on developing cells depended on the concentration of GlcN added; the addition of low concentrations of GlcN resulted in enhancement of sporulation, while higher concentrations resulted in the inhibition of sporulation. The ultrastructure of GlcN-induced spores resembled that of spores induced by the exogenous addition of glycerol, in contrast to spores isolated from mature fruiting bodies. A model by which GlcN may regulate both lysis and sporulation is presented.     

Changes in enzyme activity during differentiation in Chlamydomonas reinhardtii

The patterns of alanine dehydrogenase, glutamate dehydrogenase and malate dehydrogenase activity were studied during the normal vegetative cell cycle and during the process of gametic differentiation and dedifferentiation in synchronized cultures of Chlamydomonas reinhardtii. During all three phases of growth and differentiation the synthesis of DNA was also measured. During gametic differentiation all three enzyme levels were suppressed compared to vegetative cells although DNA and cell number were comparable. During gametic dedifferentiation no DNA synthesis occurred during the first 24 h cycle and only a doubling during the second. It was not until the third cycle that a normal 4-fold increase in DNA was observed. Cell number followed a similar pattern. Athough the levels of alanine dehydrogenase and malate dehydrogenase were uniformly low during the first cycle when glutamate dehydrogenase increased 4-fold, during the second cycle the patterns of these enzymes changed markedly. The enzymes did not attain levels characteristic of vegetative cells until the third cycle.     

Transfer Ribonucleic Acid Methylases During the Germination of Neurospora crassa

Transfer ribonucleic acid (tRNA) methylases were studied during the germination of spores in Neurospora crassa. The total methylase capacity and base specific tRNA methylase activities were determined in extracts from cells harvested at various stages of germination. Germinated conidia have a 65% higher methylase capacity than ungerminated conidia. Three predominant methylase activities were found in the extracts, and the relative amount of each activity was different at the various stages. Enzymes from vegetative cells catalyzed significant hypermethylation of tRNA from conidia, whereas conidial enzymes were much less active on tRNA from vegetative cells. The results indicate differences in the tRNA methylase content and tRNA species of conidia and vegetative cells.     

Mannitol Metabolism in Lentinus edodes, the Shiitake Mushroom

Mannitol metabolism was evaluated in fruiting bodies of Lentinus edodes. Cell extracts were prepared from fruiting bodies, and key enzymes involved in mannitol metabolism were assayed, including hexokinase, mannitol dehydrogenase, mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, and fructose-6-phosphatase. Mannitol dehydrogenase, fructose-6-phosphatase, mannitol-1-phosphatase, and hexokinase activities were found in extracts of fruiting bodies. However, mannitol-1-phosphate dehydrogenase activity was not detected. Mycelial cultures were grown in an enriched liquid medium, and enzymes of the mannitol cycle were assayed in cell extracts of rapidly growing cells. Mannitol-1-phosphate dehydrogenase activity was also not found in mycelial extracts. Hence, evidence for a complete mannitol cycle both in vegetative mycelia and during mushroom development was lacking. The pathway of mannitol synthesis in L. edodes appears to utilize fructose as an intermediate.     

Involvement of cyclic AMP cell surface receptors and G-proteins in signal transduction during slug migration of Dictyostelium discoideum.

The presence of G-proteins, interacting with cAMP surface receptors, was investigated in vegetative cells, aggregation-competent cells, and migrating slugs of Dictyostelium discoideum. Our results indicate that G-proteins are present in all stages. In vegetative cells there is a limited number of cAMP receptors but no effect of GTP tau S on cAMP binding could be detected; in addition, no effect of cAMP on GTP tau S binding or GTPase activity was observed. In both aggregation-competent cells and slugs GTP tau S inhibits cAMP binding, while cAMP stimulates GTP tau S binding and high-affinity GTPase. Since the presence of G-proteins coupled to cAMP receptors could be demonstrated in slugs, the involvement of the effector enzymes adenylate cyclase and phospholipase C was investigated. The results show that adenylate cyclase activity is stimulated by GTP tau S in both stages and that in cells from migrating slugs the Ins(1,4,5)P3 production is increased upon stimulation with cAMP. The possible involvement of G-proteins in signal transduction during the slug stage of D. discoideum is discussed.     

Developmental changes in the modification of lysosomal enzymes in Dictyostelium discoideum

Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes, N-acetylglucosaminidase, beta-glucosidase, alpha-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of beta-glucosidase, alpha-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and alpha-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins.     

Analysis of Airborne Actinomycete Spores with Fluorogenic Substrates

The reactions between seven fluorogenic substrates and different groups of enzymes, esterases, lipases, phosphatases, and dehydrogenases, were studied in a search for a new method for the detection of actinomycete spores. Fluorescence measurement was chosen as a fast and sensitive method for microbial analysis. The focus of the research was on the spores of important air contaminants: Streptomyces albus and Thermoactinomyces vulgaris. For the measurement of the enzymatic activity, the chosen fluorogenic substrate was added to a mixture of spores and nutrient media, and the resulting fluorescence was measured with a spectrofluorometer. Fluorogenic substrates were found to show enzymatic activities even for dormant spores. Comparison of the enzymatic activities of dormant spores with those of vegetative cells showed similarity of the enzymatic profiles but higher activity for vegetative cells. The increase of enzymatic activity from dormant spores to vegetative cells was not linear but fluctuating. The largest fluctuations were found after 4 to 5 h of incubation. The enzymatic activities of S. albus were 10 to 50 times lower than those of T. vulgaris, except for the dehydrogenase activity, which was seven times higher. These results indicate that analysis with fluorogenic substrates has the potential for becoming a fast and sensitive method for the enumeration and identification of airborne actinomycete spores.     

De novo purine synthesis in vegetative cells and myxospores of Myxococcus xanthus

This study was designed to determine whether vegetative cells and myxospores of Myxococcus xanthus were capable of classical de novo purine biosynthesis. To answer this question, vegetative and myxospore extracts of M. xanthus FBa were tested for their ability to synthesize the second de novo intermediate, 5'-phosphoribosylglycinamide, from beginning precursors either by way of phosphoribosyl-pyrophosphate amido transferase (EC 2.4.2.14) or ribose-5-phosphate amino transferase. Both the amido and amino transferase routes occurred in both types of extracts, and both enzymes appear to be present at about the same level (per milligram of protein) in vegetative cells, myxospores, and in a bacterial prototype, Salmonella typhimurium. The dose response of the vegetative and myxospore forms of both enzymes towards adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) suggests that the allosteric structure of both enzymes is changed little by sporulation. Both enzymes were inhibited to varying degrees by a variety of purine nucleotides besides AMP, GMP, and 3':5' cyclic AMP.     

Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases

LytF, LytE, and LytC are vegetative cell wall hydrolases in Bacillus subtilis. Immunofluorescence microscopy showed that an epitope-tagged LytF fusion protein (LytF-3xFLAG) in the wild-type background strain was localized at cell separation sites and one of the cell poles of rod-shaped cells during vegetative growth. However, in a mutant lacking both the cell surface protease WprA and the extracellular protease Epr, the fusion protein was observed at both cell poles in addition to cell separation sites. This suggests that LytF is potentially localized at cell separation sites and both cell poles during vegetative growth and that WprA and Epr are involved in LytF degradation. The localization pattern of LytE-3xFLAG was very similar to that of LytF-3xFLAG during vegetative growth. However, especially in the early vegetative growth phase, there was a remarkable difference between the shape of cells expressing LytE-3xFLAG and the shape of cells expressing LytF-3xFLAG. In the case of LytF-3xFLAG, it seemed that the signals in normal rod-shaped cells were stronger than those in long-chain cells. In contrast, the reverse was found in the case of LytE-3xFLAG. This difference may reflect the dependence on different sigma factors for gene expression. The results support and extend the previous finding that LytF and LytE are cell-separating enzymes. On the other hand, we observed that cells producing LytC-3xFLAG are uniformly coated with the fusion protein after the middle of the exponential growth phase, which supports the suggestion that LytC is a major autolysin that is not associated with cell separation.     

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

Studies on the Induction of Reversible Hydrogenase from Cyanobacterium Anabaena variabilis and its Properties

The reversible hydrogenase in vegetative cells of A. variabilis cultured on NH4+ or N-free medium was induced by sparging with N2 for 24 hours under light. Both anaerobic condition and illumination appear to be necessary for the induction of hydrogenase in this algae. The properties of the hydrogenase in cell-free extract obtained from the cells grown on two nitrogen sources are similar: (1) Both the enzymes are able to evolve H2 in the presence of reduced methyl viotogen as electron donor, and to uptake H2 in the presence of benzyl viologen as electron acceptor. (2) The enzymes posses the thermal stability and are stable to O2. (3) The optimum pH required for H2 evolution activity of the enzymes is 7.0–7 5. (4) The Km of the enzymes obtained from NH4+ grown cells and N-free grown cells is 300 mmol/l and 295 mmol/l, respectively. So the high Km measured here suggests that the enzymes in both cases function physiologically as H2 evolution. (5) The activities of both enzymes are inhibited by CO but are not affected by C2H2. The induced H2 evolution activity of the reversible hydrogenase in cells grown on NH4+ reached 1530 nmol H2/mg dry wt, h, which was 3 to 5 times higher than from cells grown on N-free medium. Our experiment results indicate that the appearance of heterocysts of A. variabilis cultured on N-free medium affects the synthesis of reversible hydrogenase and the regulation of its activity.