Construction of a shuttle vector betweenEscherichia coli andZymomonas anaerobia

Summary A 1.7-kb cryptic plasmid was isolated fromZymomonas anaerobia and used to construct a shuttle vector inserting useful parts of pUC9, pBR322, and pRK2501.Escherichia coli was employed to clone the new plasmid designated pSR12. The 7.7-kb plasmid pSR12 reisolated from the host cells could transform competent cells ofZ.anaerobia at 2×10^–7 frequency. This shuttle vector contains two antibiotic resistance markers, Kan^r and Tet^r, as well as restriction sites such as EcoRI, PstI, and XhoI, suitable for DNA recombinations.     

Enzymatic hydrolysis of penicillin V to 6-aminopenicillanic acid byFusarium oxysporum

Summary A strain ofFusarium oxysporum was identified as having an intracellular penicillin V acylase activity (penicillin V amidohydrolase EC 3.5.1.11). Activity was induced by phenoxyacetic acid and had a good tolerance for high substrate and product concentrations. Washed cells could be used repeatedly for the complete hydrolysis of 5% penicillin V solutions. The enzyme was partially purified and concentrated from disrupted cells by fractional precipitation with water miscible solvents.     

DENN domain-containing protein FAM45A regulates the homeostasis of late/multivesicular endosomes

DENN (differentially expressed in normal cells and neoplasia) domain-containing proteins are a family of guanine nucleotide exchange factors (GEFs) for Rab small GTPases and coordinate a plethora of intracellular membrane trafficking events. FAM45A is a non-classical DENN domain protein, whose function was unknown. In this study, we characterized cellular roles of FAM45A. We found that FAM45A localized mainly in late/multivesicular endosomes. Depletion of FAM45A resulted in clustering of endosomes to the perinuclear region. The endocytosis of EGF receptor was impaired in FAM45A knockdown cells due to a delay in the early-to-late endosome transition. Furthermore, the secretion of selected exosome subpopulations was also attenuated in FAM45A knockdown cells. Consistent with these results, Rab27a and Rab27b, two Rabs involved in endosome motility and exosome biogenesis, were found to act downstream of FAM45A pathway. FAM45A colocalized with Rab27a/b and formed complex with them in a nucleotide-dependent manner. Taken together, FAM45A defines a novel regulatory step in the homeostasis of late endocytic pathway, including endosomal positioning, maturation and secretion, possibly through activating Rab proteins such as Rab27a/b.     

Immobilization of yeasts on titanium activated inorganic supports

Summary A study of the immobilization of yeast cells with invertase activity by the metal link method was performed. Baker's yeast cells were immobilized on titanium activated porous silica support and on its alkylamine and aldehyde derivatives, their initial activities being 19.6, 39.9 and 10.6 U/ml of reactor respectively. When crosslinking of the immobilized cells was performed, an initial activity of 48.2 U/ml was achieved on the titanium activated support. Batch long-term stability tests were car ried out for 400 hours and the crosslinked preparations showed an unsta ble behaviour compared with the very stable preparations obtained with the simple metal-link method.A higher activity (56.2 U/ml) was obtained when a titanium activated macroporous support, pumice stone, was used as cell carrier, which compared favourably with calcium alginate entrapped cells (17.7 – 31.3 U/ml)     

Correlation between binding of Agrobacterium tumefaciens by root cap cells and susceptibility of plants to crown gall

We compared the binding of Agrobacterium tumefaciens by freshly isolated root cap cells with susceptibility of plants to crown gall tumorigenesis. A high binding reaction was strongly correlated with susceptibility to tumorigenesis in a survey of the binding of strain B6 to cells from 48 species in 17 families. In reciprocal experiments with nine virulent A. tumefaciens strains, tumors developed in plant-bacteria combinations that gave a high binding response in the root cap cell assay. Binding was quantified by direct measurement of the number of bacteria bound to the periphery of individual cells. Root cap cells from six susceptible species bound significantly more bacteria than did cells from five resistant species.     

Enzymic synthesis of o-succinylbenzoyl-CoA in cell-free extracts of anthraquinone producing Galium mollugo L. cell suspension cultures

o-Succinylbenzoic acid (OSB) is an intermediate in the biosynthesis of shikimatederived anthraquinones. The cell free activation of o-succinylbenzoic acid in extracts of anthraquinone producing cells of Galium mollugo L. is demonstrated for the first time. This activation depends on the presence of ATP, coenzyme A and Mg²⁺. The o-succinylbenzoic acid coenzyme A ester was identified by converting it to 1,4-dihydroxy-2-naphthoic acid by a bacterial enzyme, viz. naphthoatesynthase. It is thus demonstrated that the o-succinylbenzoic acid coenzyme A ester derived from bacteria and from Galium mollugo cells are identical.     

A new estimation method for plant cell viability by determining electron transport activity

A new method with which to estimate the viability of tissue-cultured plant cells was developed. In this method, electrons from the electron transport system of Coptis japonica cells were trapped by artificial electron acceptors, and the color of the reduced acceptor was monitored with a spectrophotometer through an optical fiber as surface-reflected light. Cell viability is represented by the amount of increased reflected light per unit time as electron transport activity (ETA). The electron transport activities of cultured Coptis japonica cells that had been effected in viability by the addition of different concentrations of a microbial broth, were related to the ability of the cells to proliferate. When various microbial broths were added to our Coptis japonica cultures, there was a negative correlation between electron transport activity and the amount of berberine released. During usual subculture, electron transport activity increased from the onset of culture, reached a maximum in the late log phase, then decresed rapidly.     

Selective production of phenylalanine from phenylpyruvate using growing cells ofCorynebacterium glutamicum

Summary Phenylalanine was produced from phenylpyruvate by growing cells of a mutant strain ofCorynebacterium glutamicum over-producing valine. A defined medium was used to minimize the accumulation of valine. The maximum phenylalanine concentration achieved was 44.5 mM (7.5 g/l) with a phenylpyruvate molar conversion of 75%.     

Limitations on the use of immobilized bacterial biofilms for enzymically-mediated uranyl ion accumulation

Summary A phosphatase-overproducing mutant of a Citrobacter sp. was evaluated in terms of its ability to self-immobilize as a biofilm on reticulated foam supports and to accumulate uranium when retained in a flow-through column. Parallel studies using polyacrylamide gel-immobilized cells suggested decreased substrate was available to cells of the mutant.     

Protoplast formation and cell wall regeneration inClostridium thermocellum

Summary A protocol was developed for the production ofClostridium thermocellum protoplasts and the regeneration of protoplasts into vegetative cells. Protoplasts were prepared by exposure of whole cells to lysozyme (125 ug/ml for 10 min. at 45 C). Protoplasts plated on regeneration agar reverted to walled cells with a frequency of up to approximately 0.3 % of input cells.     

Optimization of growth conditions for the induction of tyrosine decarboxylase inStreptococcus faecalis

Summary Growth conditions were investigated for optimal tyrosine decarboxylase (EC 4.1.1.25) activity in acetone dried cells ofStreptococcus faecalis. A growth pH of 6.0 was found optimal for enzyme induction. The enzyme was also shown to be growth-associated which indicates that continuous fermentation is preferable for optimal process productivity.     

Detection of bacterial chitinase activity in transformed plant tumour cells using a specific exochitinase substrate

Methods for the detection of bacterial chitinase activity were compared. The soluble substrate p-nitrophenyl-ß-D-N,N diacetyl chitobiose (NDC) was more sensitive in detecting purified chitinase of Serratia marcescens than assays measuring degradation of a solid chitin substrate by either radiochemical or colorimetric means. A chimaeric gene containing a S. marcescens chitinase gene under control of a Cauliflower Mosaic Virus 35S promoter and nopaline synthase terminator sequences was constructed and transferred to tobacco tumour cells using Agrobacterium tumefaciens as a vector. The rate of hydrolysis of the NDC substrate was three fold greater with cell extracts of both pooled and individual tumours carrying the chimaeric chitinase gene than in control tumours. It was calculated from the enzyme activity data that the foreign bacterial chitinase contributed 0.1% of the total soluble protein in transformed plant cells. This level of expression of this gene was not detectable using the less sensitive assays employing solid chitin substrate. These results indicate that NDC is a preferable substrate for assaying bacterial chitinase in transformed plant cells.     

pH influence on ethanol production and retained biomass in a passively immobilizedZymomonas mobilis system

Summary A broad pH range of 4.5–7.5 for maximum ethanol productivity and ethanol yield was observed with a passively immobilizedZ. mobilis system. Total retained biomass (as suspended flocs and entrapped cells) was >50 g/l for medium pH values between 4.0–8.0. The entrapped cells to suspended flocs ratio was highest at pH 4.0, whereas at pH above 5.2 it was close to 1.0. The observed enhancement of cell immobilization on the packing support at low pH seemed to be related to formation of bacterial filaments.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

A two-layer culture method was established that uses an organic solvent to remove shikonin derivatives produced on cell surfaces during the culture of suspension cells of Lithospermum erythrorhizon. Some paraffins and a fatty acid ester made suitable solvents, whereas olefins and aromatic solvents extensively inhibited the production of shikonin derivatives. The yield of derivatives increased with an increase in the carbon chain length of the n-paraffin used as the solvent and when the oxygen supply was sufficient it reached the value found for the ordinary culture method.     

An automatic,on-line glucose analyzer for feed-back control of fed-batch growth of Escherichia coli

Summary A computer-assisted on-line glucose analyzer was developed for feed-back control of cell growth. Using this system the glucose consumption rate for Escherichia coli was determined to be linear during batch culture at 0.37 g/hr. On-line feed-back control of glucose concentration at 1.5±0.5 g/L was used with fed-batch cultures to produce 31.2 g dry weight of E. coli cells/L in 12 h.     

Ethanol fermentation of hexose and pentose wood sugars produced by hydrogen-fluoride solvolysis of aspen chips

Summary Hexose and pentose sugars, produced by hydrogen-fluoride solvolysis of aspen wood chips, were totally consumed in a coculture fermentation by Zymomonas mobilis and a mutant of Clostridium saccharolyticum. Z. mobilis converted the glucose to ethanol, while the mutant, which was improved in both ethanol production and tolerance, converted the xylose component to ethanol. A high conversion efficiency of wood sugars to ethanol was obtained, and the cells after the fermentation were successfully used for cell recycle.NRCC no. 23211     

A novel method to produce Anabaena-free Azolla by in vitro fertilization of micromanipulated megasporocarps

In the Azolla-Anabaena azollae symbiotic system, Anabaena akinetes get entrapped between the indusium and the apical cap of the megaspore apparatus during megasporocarp development, thus maintaining the continuity of the cyanobacterial association throughout the life cycle of the fern. The entrapped akinetes serve as the source of inoculum for infecting the new sporophyte when it is emerging from the megaspore apparatus. A procedure to generate Anabaena-free Azolla was developed by fertilizing the germinating megasporocarps in which the indusium along with the akinetes were removed by micromanipulation. This method has the advantage of not requiring drastic treatments of Azolla with antibiotics to eliminate the endosymbiotic cyanobacterial cells. Details of this new method and its usefulness in studies aimed at recombination of Azolla with Anabaena azollae are discussed.Abbreviations IRRI International Rice Research Institute - I^– IRRI medium devoid of combined nitrogen - I⁺ IRRI medium containing combined nitrogen - SDS sodium dodecyl sulfate     

Inhibition of cultured cell growth by tungstate and molybdate

The growth of suspension cultured cells of Nicotiana tabacum (tobacco) was inhibited completely by 100 M tungstate. Even though molybdate reversed the tungstate inactivation of nitrate reductase activity, the growth inhibition was not reversed. The growth inhibition of N. tabacum, Daucus carota, Glycine max and Solanum tuberosum suspension cultured cells by tungstate was similar in media with or without amino acids as a source of reduced nitrogen. Only in the case of G. max was a slight reversal caused by the amino acids. Tungstate was slightly less inhibitory to the growth of a nitrate reductase-lacking mutant N. tabacum line (nia-63) than to the line with nitrate reductase. These results indicate that tungstate must inhibit the cell growth of the four species used, predominantly, in some way other than by inhibiting nitrate reductase activity. Similar studies with molybdate, a sulfate analog which apparently competes with sulfate at the ATP sulfury-lase enzyme, showed that 1 mM concentrations were completely inhibitory to cell growth. The addition of sulfate or cysteine, as a source of reduced sulfur, and amino acids, as a source of reduced nitrogen, in most cases did not reverse the molybdate inhibition appreciably. Some reversal was seen only by sulfate with D. carota cells and by cysteine plus amino acids with D. carota and G. max. These results indicate that selection for tungstate or molybdate resistance will in general not select for higher levels or other alterations in the activity of nitrate reductase or ATP sulfurylase, respectively, since these ions do not inhibit growth by primarily affecting these enzymatic steps in cultured cells of the four species studied.     

Metalloprotease ADAMTS-1 decreases cell migration and invasion modulating the spatiotemporal dynamics of Cdc42 activity

ADAMTSs (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are secreted proteases dependent on Zn²⁺/Ca²⁺, involved in physiological and pathological processes and are part of the extracellular matrix (ECM). Here, we investigated if ADAMTS-1 is required for invasion and migration of cells and the possible mechanism involved. In order to test ADAMTS-1's role in ovarian cancer cells (CHO, NIH-OVCAR-3 and ES2) and NIH-3 T3 fibroblasts, we modified the levels of ADAMTS-1 and compared those to parental. Cells exposed to ADAMTS-1-enriched medium exhibited a decline in cell migration and invasion when compared to controls with or without a functional metalloproteinase domain. The opposite was observed in cells when ADAMTS-1 was deleted via the CRISPR/Cas9 approach. The decline in ADAMTS-1 levels enhanced the phosphorylated form of Src and FAK. We also evaluated the activities of cellular Rho GTPases from cell lysates using the GLISA® kit. The Cdc42-GTP signal was significantly increased in the CRISPR ADAMTS-1 ES-2 cells. By a Förster resonance energy transfer (FRET) biosensor for Cdc42 activity in ES-2 cells we demonstrated that Cdc42 activity was strongly polarized at the leading edge of migrating cells with ADAMTS-1 deletion, compared to the wild type cells. As conclusion, ADAMTS-1 inhibits proliferation, polarization and migration.     

Alkaloid production in cell suspension cultures of Thalictrum flavum and T. dipterocarpum

Both cell suspension cultures of Thalictrum flavum and T. dipterocarpum were found to produce berberine (0.3 and 0.4 g/l, respectively) as a main alkaloid. Berberine production in the latter was markedly stimulated by 1-naphthaleneacetic acid in combination with 6-benzylaminopurine, whereas it was rather suppressed by the same auxin in the former. T. flavum cultures accumulated berberine and columbamine in the cells without releasing them into medium. On the other hand, T. dipterocarpum cultures released berberine into medium during the logarithmic growth phase, but thereafter accumulated all the berberine synthesized in the cells.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - TFG a culture strain of T. flavum ssp. glaucum - TDP a culture strain of T. dipterocarpum