Antioxidative enzymes of sulfate-reducing bacterium desulfovibrio desulfuricans: superoxide dismutase and peroxidases

Extracts of Desulfovibrio desulfuricans B-1388 cells grown under anaerobic conditions displayed superoxide dismutase activity. The maximal activity was found during the stationary growth phase. The enzyme was virtually completely located in the periplasm fraction. D. desulfuricans B-1388 lacked catalase activity but contained active NADH- and NADPH-peroxidases. The activity of NADH-peroxidase depended on the physiological state of the culture. On changing the growth conditions (the presence of 5% CO in the gaseous phase), the activity of superoxide dismutase decreased.     

Rearrangement of enzyme patterns in maize callus and suspension cultures

The development of enzyme patterns was followed in the course of: (a) the irreversible cell differentiation via division and expansion to maturity in the root tip and coleoptile of the intact seedlings, (b) the irreversible cell dedifferentation associated with induction and establishment of callus from the growing internodes, and (c) the growth cycle (proliferationstationary phase) in callus and cell-suspension cultures of maize (Zea mays L.). By measuring the activities of glycolytic, mitochondrial, microbody and hydrolytic enzymes cells proliferating in vivo and in vitro could be compared and changes related to cessation or resumption of cell division could be studied.Proliferating cells of callus and suspension cultures maintained by serial culture did not differ from those of the root meristem and coleoptile in the specific activities of hexokinase, phosphoglycerate kinase and phosphopyruvate hydratase. Proliferation in vitro resulted in an enormous increase in the ratio g glutamate-dehydrogenase/cytochrome-oxidase activity and in the level of acid-phosphatase activity, with concomitant drop in galactosidase and xylosidase activity. A 3-5-fold increase of alcohol-dehydrogenase, lactate-dehydrogenase and catalase activities was characteristic of dividing callus cells, while a ca. 100-fold increase in the fructofuranosidase-to-glucosidase activity ratio marked cell proliferation in suspension-cultured cells.Changing enzyme activities after cessation of proliferation were quite similar in root tips and coleoptiles, except those of alcohol dehydrogenase and catalase. The enzyme rearrangement during callus establishment and in the growth cycle of callus cultures was in most cases comparable to that in the intact tissues, while the changes from the dividing to the non-dividing cells in suspension cultures, in contrast, differed widely from those in the intact tissues and callus. Galactosidase and xylosidase were the only activities that showed a similar trend of changes in all the investigated, intact and in-vitro-grown cells.Thus, judged by the pattern of enzyme development, the cell suspension appears to be a unique system, virtually unrelated to the growing cells of the intact tissues. It is also very difficult to draw a definite distinction between the metabolic consequences of cell growth and enzyme modulations in cell suspensions as the cells adapt their metabolism to the environmental changes in liquid medium.     

Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.     

Periplasmic superoxide dismutases in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 cells cultured microaerobically (dissolved O₂ tension 1% of saturation), expressed proteins with superoxide dismutase (SOD) activity. The majority (roughly 95%) of total cell superoxide dismutase activity was located in the cell periplasm with little or no activity in the cell cytoplasm. Irontype SOD (FeSOD) contributed 88% of the total activity activity detected, although a manganese-type SOD (MnSOD) was present in the periplasm as well. Cells cultured at a higher dissolved O₂ tension (10% of saturation) expressed increased activity of the MnSOD relative to that of the FeSOD.     

Location of anaerobic respiratory enzyme trimethylamine N-oxide reductase in the cytoplasmic membrane ofEscherichia coli strain K10

Trimethylamine N-oxide (TMAO) reductase, which is anaerobically induced by TMAO, is a terminal enzyme in anaerobic electron transport inEscherichia coli. When the organism was anaerobically grown with TMAO, a marked increase in the specific activity of TMAO reductase was observed mainly in a cell membrane fraction and stopped after exhausting TMAO. On the other hand, activity was moderately increased in a soluble fraction of the cell even after exhaustion of TMAO. Immunoblot analysis with an antiserum against the TMAO reductase purified from the soluble fractions showed that the cells growing with TMAO contained only a membrane-bound enzyme, which has a molecular mass of 94 kDa, while a soluble enzyme with 92 kDa appeared in the stationary growth phase lacking TMAO. Experiments with right-side-out and inside-out vesicles of cytoplasmic membrane indicated that the membrane-bound enzyme faces the cytoplasm. The soluble enzyme was mainly found in the cytoplasm of the cell, but also at a negligible amount in the periplasm. The membrane-bound form of TMAO reductase functioning in anaerobic electron transport seems to be cleaved and released into the cytoplasm as soluble enzyme after exhaustion of TMAO.     

Intracellular peptidoglycan hydrolases of the bacterium Xanthomonas campestris XL-1

A system of intracellular peptidoglycan hydrolases of Xanthomonas campestris XL-1 comprises about 10 enzymes of different localization and substrate specificity. Seven enzymes (A₁-A₇) are localized in cytosol, one enzyme (A₈) in periplasm, and two enzymes (A₉, A₁₀) were found in the fraction of cell walls and membranes. While the culture is entering the logarithmic growth stage from the stationary stage, a change occurs in the activity of the cytosolic enzymes: A₁ significantly increases, and A₅ and A₆ decrease. The spectrum of cytosolic enzymes also depends on the growth medium composition. The enzyme A₇ present in cells secreting extracellular enzymes (medium 5/5) was not found in non-secreting cells (LB medium). Unlike extracellular enzymes, intracellular peptidoglycan hydrolases are primarily acidic proteins. The data indicate that the system of intracellular peptidoglycan hydrolases of X. campestris is under complex and strict regulation.     

Characterization of a Cell Membrane-Associated Proteinase from Lactobacillus helveticus CRL 581

The production of a proteinase from Lactobacillus helveticus CRL 581 was studied. The highest specific activity was found at the early exponential growth phase of cells cultured in milk. The lowest levels of proteinase were detected in MRS broth, while in the casein–yeast extract–glucose broth enzyme production increased gradually during the fermentation and reached maximal values at the stationary phase. The proteinase, found to be associated with the cell membrane fraction, hydrolyzed β-casein more rapidly than α-casein. The enzyme was not released from washed cells in the presence or absence of calcium, which suggests that the enzyme did not undergo self-digestion. Received: 28 January 1997 / Accepted: 8 March 1997     

Initial characterization of two extracellular autolysins from Pseudomonas aeruginosa PAO1.

Two extracellular autolysins have been detected in the spent culture supernatants of Pseudomonas aeruginosa PAO1 by using renaturing polyacrylamide gel electrophoresis. The two autolysins were isolated from the culture supernatant by trichloroacetic acid precipitation and were shown to have apparent molecular masses of 26 and 29 kDa. The 26-kDa autolysin first appears during the early exponential phase of growth and then declines sharply, while the 29-kDa autolysin first appears in the late exponential phase of growth and continues well into the stationary phase. Fractionation of whole cells indicated that the 26-kDa enzyme was also localized within the periplasm, with a lesser amount of activity associated with the cytoplasmic membrane. The 29-kDa autolytic activity was distributed within the cell equally between the periplasm and the cytoplasmic membrane. The pH optima of the isolated 26- and 29-kDa autolysins are 6.0 and 5.0, respectively. Further evidence from both protease susceptibility and inhibition studies confirms that these two extracellular autolysins isolated from P. aeruginosa PAO1 are separate and distinct.     

Oxidative Stress and Antioxidant Cell Protection Systems in the Microaerophilic Bacterium Spirillum winogradskii

The influence of oxygen availability during cultivation on the biosynthetic processes and enzymatic activities in the microaerophilic bacterium Spirillum winogradskii D-427 was studied, and the roles played by different systems of the defense against oxidation stress were determined. The metabolic adjustments caused by transition from microaerobic (2% O₂) aerobic conditions (21% O₂ of the gas phase) were found to slow down constructive metabolism and increase synthesis of exopolysaccharides as a means of external protection of cells from excess oxygen. This resulted in a twofold decline of the growth yield coefficient. Even though the low activity of catalase is compensated for by a multifold increase in the activities of other cytoplasmic enzymes that defend against toxic forms of O₂—peroxidase and enzymes of the redox system of glutathione (glutathione peroxidase and glutathione reductase)—massive lysis of cells starts in the midexponential phase and leads to culture death in the stationary phase because of H₂O₂ accumulation in the periplasm (up to 10 g/mg protein). The absence in cells of cytochrome-c-peroxidase, a periplasmic enzyme eliminating H₂O₂, was shown. It follows that the major cause of oxidative stress in cells is that active antioxidant defenses are located in the cytoplasm, whereas H₂O₂ accumulates in the periplasm due to the lack of cytochrome-c-peroxidase. The addition to the medium of thiosulfate promotes elimination of H₂O₂, stops cell lysis under aerobic conditions, lends stability to cultures, and results in a threefold increase in the growth yield.     

Multi-parameter flow cytometry as a tool to monitor heterotrophic microalgal batch fermentations for oil production towards biodiesel

Multi-parameter flow cytometry was used to monitor cell intrinsic light scatter, viability, and lipid content of Chlorella protothecoides cells grown in shake flasks. Changes in the right angle light scatter (RALS) and forward angle light scatter (FALS) were detected during the microalgal growth, which were attributed to the different microalgal cell cycle stages. The proportion of cells not stained with PI (cells with intact cytoplasmic membrane) was high (> 90%) during the microalgal growth, even in the latter stationary phase, suggesting that the microalgal cells built-up storage materials which allowed them to survive under nutrient starvation, maintaining their cytoplasmic membranes intact. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional lipid extraction method was found for this microalga, making this method a suitable and quick technique for the screening of microalgal strains for lipid production, optimization of biofuel production bioprocesses, and scale-up studies. The highest oil content (∼28% w/w dry cell weight, estimated by flow cytometry) was observed in the latter stationary phase. In addition, C. protothecoides oil also depicted the adequate fatty acid methyl ester composition for biodiesel purposes at this growth phase, suggesting that the microalgal oil produced during the latter stationary phase could be an adequate substitute for diesel fuel. Medium growth optimization for enhancement of microalgal oil production is now in progress, using the multi-parameter approach.     

Staphylolytic enzyme from Pseudomonas aeruginosa: characterization and immunocytochemical localization

Staphylolytic enzyme, a specific peptidase produced by Pseudomonas aeruginosa, has been characterized by using immunochemical procedures. Lytic activity was detected in the extracellular medium of Pseudomonas cultures at the beginning of the stationary growth phase. No activity was detected in bacterial cells. However, lytic protein antigen was present in periplasmic and cytoplasmic fractions, suggesting that staphylolytic enzyme is synthesized as an inactive precursor which becomes active during translocation to the extracellular broth. Results obtained in immunolocalization experiments indicate the presence of the precursor in the outer part of cells. The export pathway of staphylolytic enzyme through the periplasmic space is proposed.Abbreviations DCE dialyzed crude extract - CFU colonies forming units - LU lytic unit     

Characteristics of copper tolerance in Yarrowia lipolytica

We discovered that a mutant strain of the dimorphic yeast Yarrowia lipolytica could grow in the yeast form in high concentrations of copper sulfate. The amount of metal accumulated by Y. lipolytica increased with increasing copper concentrations in the medium. Washing with 100 mM EDTA released at least 60% of the total metal from the cells, but about 20–25 μmol/g DW persisted, which represented about 30% of the soluble fraction of cultured cells. The soluble fraction (mainly cytosol) contained only about 10% of the total metal content within cells cultured in medium supplemented with 6 mM copper. We suggest that although a high copper concentration induces an efflux mechanism, the released copper becomes entrapped in the periplasm and in other parts of the cell wall. Washing with EDTA liberated not only copper ions, but also melanin, a brown pigment that can bind metal and which located at the cell wall. These findings indicated that melanin participates in the mechanism of metal accumulation. Culture in medium supplemented with copper obviously enhanced the activities of Cu, Zn-SOD, but not of Mn-SOD.     

Thymidine kinase activity in mouse embryo fibroblast cells infected with murine cytomegalovirus

Thymidine kinase activity was found in whole cell extracts of growing and stationary mouse embryo fibroblast cells after infection with murine cytomegalovirus. Determination of the kinetic constants and heat stability characteristics indicated that the enzyme activity from infected cells was different to that found in uninfected cells in the growth phase. The expression of thymidine kinase activity during virus replication was reflected by the incorporation of (6-³H) thymidine into acid precipitable fractions of infected cell cultures. Preliminary data from kinetic studies showed a reduction in the phosphorylation of thymidine by this enzyme activity in the presence of Acyclovir, a potent inhibitor of herpes virus replication.     

State of water in extremely halophilic bacteria: Heat of dilution ofHalobacterium halobium

Summary Heat of dilution ofHalobacterium halobium was measured when thick pastes of the bacteria, harvested throughout a complete growth cycle, were lysed by mixing with 40 times their volume of water in a microcalorimeter. A series of comparative measurements was made with pastes of bacteria previously disrupted by freezing and thawing but otherwise identical to the pastes of whole bacteria. The frozen-thawed pastes gave endothermic values some 18% greater than those obtained with intact bacteria; the difference was highly significant. Evidence was obtained that the mechanical component of bursting did not contribute to the difference between whole and lysed bacteria. On the other hand, when a correction was applied for heat of mixing of intracellular salts with extracellular NaCl, such as occurs when the bacteria lyse, the difference between whole and disrupted organisms was largely eliminated from exponential phase halobacteria but not from those harvested in stationary phase. It is concluded that there is no evidence, as reflected in heat of dilution, of abnormal solution properties of the cytosol of young halobacteria, which are rich in potassium. On the other hand, and paradoxically, some doubt remains about stationary phase organisms whose cytosol has a much higher Na⁺ content (and Na/K⁺ ratio) than the cytosol of exponential phase bacteria.     

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen.

A lambda gt11 cDNA library from Candida albicans ATCC 26555 was screened by using pooled sera from two patients with systemic candidiasis and five neutropenic patients with high levels of anti-C. albicans immunoglobulin M antibodies. Seven clones were isolated from 60,000 recombinant phages. The most reactive one contained a 0.9-kb cDNA encoding a polypeptide immunoreactive only with sera from patients with systemic candidiasis. The whole gene was isolated from a genomic library by using the cDNA as a probe. The nucleotide sequence of the coding region showed homology (78 to 79%) to the Saccharomyces cerevisiae TDH1 to TDH3 genes coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and their amino acid sequences showed 76% identity; thus, this gene has been named C. albicans TDH1. A rabbit polyclonal antiserum against the purified cytosolic C. albicans GAPDH (polyclonal antibody [PAb] anti-CA-GAPDH) was used to identify the GAPDH in the beta-mercaptoethanol extracts containing cell wall moieties. Indirect immunofluorescence demonstrated the presence of GAPDH at the C. albicans cell surface, particularly on the blastoconidia. Semiquantitative flow cytometry analysis showed the sensitivity of this GAPDH form to trypsin and its resistance to be removed with 2 M NaCl or 2% sodium dodecyl sulfate. The decrease in fluorescence in the presence of soluble GAPDH indicates the specificity of the labelling. In addition, a dose-dependent GAPDH enzymatic activity was detected in intact blastoconidia and germ tube cells. This activity was reduced by pretreatment of the cells with trypsin, formaldehyde, and PAb anti-CA-GAPDH. These observations indicate that an immunogenic, enzymatically active cell wall-associated form of the glycolytic enzyme GAPDH is found at the cell surface of C. albicans cells.     

Levels of RNA polymerase activities during growth,encystment and germination ofPhysarum polycephalum

Summary Two RNA polymerases, enzyme A and enzyme B, were prepared fromPhysarum plasmodia. They are not located in the cytosol. Isolated nuclei, however, contain only a fraction of the total RNA polymerase activity: 1.5–19% depending on the nuclear preparation method.The level of RNA polymerase B shows little variation at developmental stages of growth and encystment, or in cysts and during germination, whereas RNA polymerase A displays a marked transient decrease in activity during the stationary growth phase before encystment.     

Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: An evolutionary scenario of subcellular compartmentation in Kinetoplastida

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in both the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoenzymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated that the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the trypanosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arranged genes were found which are 95% identical. The two encoded polypeptides differ in 17 residues. Their sequences are 72–77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, specific insertions, and a small carboxy-terminal extension containing the sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has undergone some mutations at positions coding for highly conserved residues of the active site and the NAD⁺-binding domain of GAPDH. Modeling of the protein's three-dimensional structure suggested that several of the substitutions compensate each other, retaining the functional coenzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by horizontal gene transfer during the evolution of the Kinetoplastida, in the lineage leading to the suborder Trypanosomatina (Trypanosome, Leishmania), after the divergence from the Bodonina (Trypanoplasma). Furthermore, the data clearly suggest that the original GAPDH of the Kinetoplastida has been compartmentalized during evolution.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) - HK hexokinase (EC 2.7.1.1) - PGI glucosephosphate isomerase (EC 5.3.1.9) - PGK phosphoglycerate kinase (EC 2.7.2.3) - PYK pyruvate kinase (EC 2.7.1.40) - TIM triosephosphate isomerase (EC 5.3.1.1) - SDS sodium dodecyl sulfate - SSC saline sodium citrate (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) - MYR millions of years Nucleotide sequence data reported in this paper have been submitted to the EMBL/Genbank/DDBJ nucleotide sequence databases under accession number X74535 Correspondence to: P.A.M. Michels     

A Β-glucan endo-hydrolase fromSchizosaccharomyces pombe and its role in cell wall growth

A Β-glucan hydrolase showing a marked specificity for Β-1,3-glucosidic linkages was found to be closely associated with the cell wall of the fission yeastSchizosaccharomyces pombe. Intact log-phase cells showed 40% of the enzyme activity measured in the cell homogenate. Cellular enzyme activity reached a maximum during the logarithmic growth phase and decreased sharply in the stationary phase. The possible role of the enzyme in cell wall extension is discussed. Issued as NRCC No. 12329 National Research Council of Canada Postdoctorate Fellow, 1968–69. The author gratefully acknowledges helpful discussions with Dr. B. F. Johnson.     

Inactivation of chitin synthase in Saccharomyces cerevisiae

The activity of chitin synthase extracted from whole cells of Saccharomyces cerevisiae shows reproducible changes during the course of batch cultivation. During exponential growth 5–10% of the enzyme occurs in the active form, whereas in the stationary phase no active enzyme can be detected. Of three yeast proteinases, A, B and C, only B is able to activate pre-chitin synthase and inactivate chitin synthase. A new model of the regulation is presented which accounts for the specific location as well as for termination of chitin synthesis during the budding cycle.These results were reported at the 4th International Symposium on Yeasts in Vienna, July 1974, and are part of doctoral thesis by A.H., University Freiburg (1974).