John L. Hanners

Photosynthetic preparation and characterization of 13C-labeled carbohydrates in agmenellum quadruplicatum

Abstract The blue-green alga Agmenellum quadruplicatum (strain PR6) has been used to prepare photobiosynthetically 13 C-labeled d -glucose, 2- O -(α- d -glucopyranosyl)-glyceric acid (glucosylglycerate), 2-hydroxy-1-(hydroxymethyl)ethyl α- d -gluco-pyranoside (glucosylglycerol), and α- d -glueopyranosyl β- d -fructofuranoside (sucrose). When grown to a cell density of 4.4 g.L -1 (dry weight) under nitrate-nitrogen limiting growth conditions for 120 h, the algal cells contained 38% of the dry-cell weight as(1 → 4)-α- d -glucan (amylose). About 1% of the dry-cell weight was glucosylglycerol, glucosylglycerate, and sucrose. Glutamate was obtained, together with carbohydrates of low molecular weight, when the cells were extracted with chloroform-methanol; d -glucose was recovered from the extracted cells by acid hydrolysis of the starch. The algae were grown by using 20 mol% [ 13 C] carbon dioxide for preparation of labeled carbohydrates and for cellular component identification by whole-cell n.m.r. spectroscopy.

Plasmid-conferred tetracycline resistance confers collateral cadmium sensitivity to E. coli cells

Abstract E. coli HB101 cells transformed to tetracycline resistance with the plasmids pMB9 or pBR322 display a 10 5 –10 6 -fold lower plating efficiency on agar containing 440 μ m CdCl 2 than nontransformed cells. When DNA is inserted into the Bam H1 site of the plasmid tet gene, or when DNA spanning the Bam H1 site is deleted, tetracycline resistance and cadmium hypersensitivity are both lost. In contrast, insertion of DNA into the ampicillin resistance gene does not affect cadmium hypersensitivity.

PQQ: Biosynthetic studies inMethylobacterium AM1 andHyphomicrobium X using specific13C labeling and NMR

Using13C labeling and NMR spectroscopy we have determined biosynthetic precursors of pyrroloquinoline quinone (PQQ) in two closely related serine-type methylotrophs,Methylobacterium AM1 andHyphomicrobium X. Analysis of the13C-labeling data revealed that PQQ is constructed from two amino acids: the portion containing N-6,C-7,8,9 and the two carboxylic acid groups,C-7′ and 9′, is derived-intact-from glutamate. The remaining portion is derived from tyrosine; the phenol side chain provides the six carbons of the ring containing the orthoquinone, whereas internal cyclization of the amino acid backbone forms the pyrrole-2-carboxylic acid moiety. This is analogous to the cyclization of dopaquinone to form dopachrome. Dopaquinone is a product of the oxidation of tyrosine (via dopa) in reactions catalyzed by monophenol monooxygenase (EC 1.14.18.1). Starting with tyrosine and glutamate, we will discuss possible biosynthetic routes to PQQ.

Transfer of copper from metallothionein to nonmetallothionein proteins in cultured cells

Copper uptake and distribution with time among cytoplasmic proteins were followed in cultured cells under several conditions: (1) CHO cells, which cannot synthesize metallothioneins, were labeled with67Cu in the presence of 100 μM ZnCl2; (2) Cdr30F9 cells, which contain some constitutive metallothionein (MT), were labeled in the absence of additional ZnCl2 and; (3) Cdr30F9 cells were labeled in the presence of ZnCl2, under which conditions they synthesized additional metallothioneins. The exogenous67Cu and ZnCl2, where present, were then removed, and the distributions of67Cu among size fractions of the cellular proteins were observed at intervals for 16 h. In addition, a culture identical to condition (3) above was also treated with 100 μM ZnCl2 during the redistribution period. The67Cu was initially resolved into three peaks by Sephadex G-75 chromatography: high molecular weight, intermediate molecular weight, and MT. The67Cu in the MT fraction decreased with at1/2 of 10–12 h. In contrast to this, generally, in cells with a higher initial67Cu bound to metallothionein, there was a progressive increase in the amount of67Cu eluting with the high- and intermediate-molecular-weight fractions. Since no other source of67Cu was available, these experiments suggest that copper stored in MT can be transferred to other proteins in these cells.

Biodegradation of paint stripper solvents in a modified gas lift loop bioreactor

Paint stripping wastes generated during the decontamination and decommissioning of former nuclear facilities contain paint stripping organics (dichloromethane, 2-propanol, and methanol) and bulk materials containing paint pigments. It is desirable to degrade the organic residues as part of an integrated chemical-biological treatment system. We have developed a modified gas lift loop bioreactor employing a defined consortium of Rhodococcus rhodochrous strain OFS and Hyphomicrobium sp. DM-2 that degrades paint stripper organics. Mass transfer coefficients and kinetic constants for biodegradation in the system were determined. It was found that transfer of organic substrates from surrogate waste into the air and further into the liquid medium in the bioreactor were rapid processes, occurring within minutes. Monod kinetics was employed to model the biodegradation of paint stripping organics. Analysis of the bioreactor process was accomplished with BIOLAB, a mathematical code that simulates coupled mass transfer and biodegradation processes. This code was used to fit experimental data to Monod kinetics and to determine kinetic parameters. The BIOLAB code was also employed to compare activities in the bioreactor of individual microbial cultures to the activities of combined cultures in the bioreactor. This code is of benefit for further optimization and scale-up of the bioreactor for treatment of paint stripping and other volatile organic wastes in bulk materials.

Informosomal and polysomal messenger RNA. Differential kinetics of polyadenylation and nucleocytoplasmic transport in Chinese hamster ovary cells.

The relative kinetics of cytoplasmic appearance and polyadenylation were determined for informosomal (ribosome-free) and polysomal (ribosome-associated) mRNAs of cultured Chinese hamster cells. Label appeared in polysomal mRNA 17--20 min and into informosomal mRNA 2--5 min after addition of radio-labelled uridine. Adenosine appeared in polysomal mRNA 4--5 min before uridine. In contrast, adenosine label preceded uridine into informosomal mRNA by less than 1 min. About one-third of newly formed informosomal and two-thirds of newly formed polysomal mRNA are poly(A+). The data indicate that newly formed informosomal mRNA cannot be simple precursor to polysomal mRNA. Further, the pronounced difference in time required for polyadenylation and cytoplasmic appearances of these messenger ribonucleoproteins suggests that there may be fundamental differences in their mode of processing.

Remediation of low-level mixed waste: cellulose-based materials and plutonium

Low-level mixed radioactive wastes containing cellulose-based materials and plutonium have been generated during various nuclear processing activities. Biological digestion of the organic- or cellulose- based material was examined as an environmentally acceptable and effective method of treatment for these and other similar wastes. Cellulase enzyme was used to initiate biodegradation prior to 90% destruction of the cellulose material by a sewage sludge consortium. Plutonium did not significantly effect the biodegradation. Bench-scale experimental data were used to design a batch treatment system. A cost and sensitivity analysis was performed to determine the optimal reactor size, materials of construction and media type. The sensitivity analysis indicated that while a 12-month treatment scenario using a carbon steel ball mill, sludge digester and vacuum thickener was the least expensive scenario evaluated on a levelized cost basis (

Biosynthesis of pyrroloquinoline quinone. II, Biosynthetic assembly from glutamate and tyrosine

800 per ton of waste degraded per month), the 12-month scenario using stainless steel construction and the alternative dewatering system offered the most cost-effective treatment alternative and better corrosion resistance (levelized cost of

Biosynthesis of pyrroloquinoline quinone. 1. Identification of biosynthetic precursors using carbon-13 labeling and NMR spectroscopy

1130 per ton per month). The dewatering system consisting of a disk centrifuge and sludge dryer is capable of doubling the sludge solids content and produce an overall waste reduction of 67%. The proposed waste treatment system offers a cost savings of up to 31% compared to conventional disposal practices.