Robert M. Pfister

Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product

Conditions for hyperexpression, in Escherichia coli, of the Bacillus thuringiensis var, kurstaki gene, cryIA9(c)73, encoding an insecticidal crystal protein, CryIA(c)73, were investigated by varying the promoter type, host cell, plasmid copy number, the second codon and number of terminators. The cryIA(c)73 gene was cloned into three E. coli expression vectors, pKK223-3 (Ptac promoter), pET-3a (P phi 10 promoter), and pUC19 (Ptac promoter). The level of cryIA(c)73 expression was measured by ELISA and compared to total cellular protein over growth periods of 24 and 48 h. Maximum expression levels of 284 microgram CryIA(C)73/ml (48% of cellular protein) were obtained in shake flasks with the Ptac promoter in E. coli JM103. Optimal conditions were found to be low-copy-number plasmid (pBR322 ori), 48 h of growth, in lon+ cells. A change of the genes second codon to AAA can improve expression by two to three fold but is undetectable in the presence of a strong E. coli promoter. The cryIA(c)73 gene product, in E. coli, formed crystals with the same lattice structure as the native crystals formed in B. thuringiensis (as visualized by electron microscopy). Bioassay results (insect toxicity and specificity) of the crystal produced in E. coli were similar to that produced in B. thuringiensis.

Effects of cadmium accumulation on growth and respiration of a cadmium-sensitive strain of Bacillus subtilis and a selected cadmium resistant mutant

The effects of cadmium on the growth and respiration of two strains of Bacillus subtilis are compared to the accumulation of Cd by viable and cyanide-killed cells, protoplasts and cell fractions of the strains. Growth and respiration of strain 1A1 were significantly inhibited at 10μg Cd2+/ml while the growth and respiration of strain 1A1R, a selected mutant of 1A1, were only slightly affected. Similarly, 1A1R protoplasts were more resistant to Cd than were 1A1 protoplasts. The differential resistance of the strains correlates with the accumulation of Cd by the two strains, with 1A1 accumulating approximately 10 times the level of Cd after a 4 h exposure to 1 μg Cd2+/ml. The distributions of Cd throughout the cells, however, were similar between strains. Based on the accumulation of Cd by cyanide-killed protoplasts, uptake of Cd by 1A1 appears to be an active process, while for 1A1R, Cd accumulation is independent of protoplast viability.

Adsorption of mixed metals and cadmium by calcium-alginate immobilized Zoogloea ramigera

SummaryZoogloea ramigera 115 cells were immobilized into beads of calcium alginate and used in air-bubbled column reactors to remove cadmium (Cd), zinc (Zn), manganese (Mn), lead (Pb), copper (Cu) and strontium (Sr) from dilute and concentrated solutions. By placing three bubble columns in sequence it was possible to achieve Cd adsorption efficiencies of 99% or greater. During ten applications of approximately 100 μg·ml-1 Cd to three reactors in sequence, the immobilized Z. ramigera adsorbed 99.9% of the metal. The efficiency of the first column decreased from 92.2% on the first day to 53.8% on the tenth day, but the overall efficiency remained high because of the other two reactors. Exposure of bubble columns to mixed metal solutions yielded similar results. Cd, Zn, Mn, Cu, and Sr were adsorbed at 95.9% or better when concentrations in the mixtures ranged from 12 to 117 μg·ml-1 per metal. Pb was the least efficiently adsorbed metal with 81.3% and 74.7% removed from solutions containing 12 and 23 μg·ml-1, Pb respectively. Recovery and concentration of the metals was possible with nitrilotriacetic acid (NTA) treatment; which desorbed >77% of the accumulated Cd from the immobilized cells. Recovery of metals from beads which had been exposed to mixed metals depended on the metal; only 5.8% of the adsorbed Sr was recovered, but 98.3% of Zn was desorbed.

Effects of pH, temperature, and Eh on the uptake of cadmium by bacteria and an artificial sediment

The report describes an investigation of cadmium adsorption by a Cd-resistant bacterium (Pseudomonas sp.) and a sediment matrix under various incubation conditions. (JMT)

Plasmids in tributyltin-resistant bacteria from fresh and estuarine waters

SummaryTwenty-six tributyltin (TBT)-resistant bacterial strains isolated from sediments were examined for the presence of plasmids. Plasmids of the size reported to carry metal resistance genes were not found in 15 of the strains, indicating that resistance does not have to be plasmid-mediated. Attempts to cure plasmid-containing strains using acridine organge, ethidium bromide, novobiocin or sodium dodecylsulfate, or by growth at elevated temperature were not successful, nor were plasmids transferred from TBT-resistant strains into TBT-sensitive organisms by electroporation. In a broth mating experiment however, plasmid pUM505, a conjugative plasmid known to encode chromium resistance inPseudomonas aeruginosa PAO1, was introduced into TBT-sensitiveBeijerinckia sp. MC-27 isolated from freshwater sediment. The TBT tolerance of theBeijerinckia sp. increased 100-fold, from 8.4 μM TBT inBeijerinckia sp. MC-27 to 840 μM TBT inBeijerinckia sp. MC-27 (pUM505) on solid medium. The plasmid was transferred at a frequency of approximately 6×10−4. TBT-resistant transconjugants grew faster in media containing TBT and lost their enhanced TBT tolerance and the plasmid upon serial transfer in medium without TBT. Spontaneous mutants of the donorP. aeruginosa lost both TBT resistance and the plasmid. Therefore, TBT resistance in bacteria can be plasmid-mediated. To our knowledge, this is the first report that resistance to a tin compound can be plasmid-mediated.

Aldrin: Removal from Lake Water by Flocculent Bacteria

Floc-forming bacteria isolated from Lake Erie adsorb and concentrate aldrin from colloidal dispersion so that the settling of the bacterial flocs removes aldrin from the water phase. Contemporary sediments forming in Lake Erie contain aldrin and could adsorb more. The sediments consist of a conglomerate floc of bacteria, diatoms, and inorganic and detrital particles. Flocculent bacteria also adsorb microparticulates, and this adsorption capacity represents a mechanism for sediment formation and for the removal of suspended particles including aldrin from the water column.

Plasmodium gallinaceum: fine structure by freeze-etch technique.

Abstract The fine structure of the obligate intracellular parasite Plasmodium gallinaceum was examined by the freeze-etching technique. The characteristics of this malarial parasite, from the early trophozoite stage to the segmenting schizont stage were studied, and structural aspects of each stage were examined. The malarial parasite was shown to have cytoplasmic and nuclear membrane characteristics which differ considerably from those of the host. Two types of discontinuities in the nuclear membranes of the parasites were noted. These were a small pore system in the inner nuclear membrane and a large pore system in the outer membrane. The cytoplasmic membranes of the parasite were more coarsely granular than those of the host. The outer parasite membrane which has been reported to be of host origin also differs considerably in structure from the erythrocyte membrane. This could be due to modifications after envelopment of the parasite, or could indicate another origin for the membrane.

Microparticulates: Isolation from Water and Identification of Associated Chlorinated Pesticides

Microparticulates suspended in lake water were collected by continuous centrifugation and either examined directly or placed on a linear sucrose gradient. Total residue as well as fractions of the centrifuged gradient were extracted with hexane and examined by gas chromatography for the presence of chlorinated hydrocarbon pesticides. Hexane extracts of total residues were also examined by thin-layer chromatography. Lindane and endrin were shown, by gas-liquid chromatography and thin-layer chromatography, to be associated with microparticles. These and other pesticides appeared to be selectively associated with microparticles of different densities, when gas-liquid chromatography was used, although concentrations were below the detection limits required for confirmation by thin-layer chromatography. Samples taken at different times from different locations in Lake Erie revealed different associations with hexane-soluble electron-capturing compounds.

ACCUMULATION OF CADMIUM BY IMMOBILIZED ZOOGLOEA RAMIGERA 115

SummaryZoogloea ramigera 115 was immobilized into beads of calcium-alginate and placed into batch air-bubbled column reactors. In the absence of any added nutrients the immobilized bacterium adsorbed Cd from solutions containing levels of 2 and 20 μg ml−1 per day, over a period of 21 and 20 days, respectively. Adsorption of Cd from solutions containing 20 μg ml−1 Cd was better than 90% for 16 days. Beads treated with Cd at 2 μg ml−1 never adsorbed less than 95% of the metal. Alginate adsorbed Cd as well, but inclusion of cells changed the effectiveness of adsorption. Of a 250 μg ml−1 Cd solution, alginate adsorbed 70.4% Cd in 60 min whereas alginate plus cells adsorbed 90.5% in the same time span. Temperature had no effect on adsorption by immobilized cells at levels of 2 and 10 μg ml−1 Cd. However at higher concentrations, binding was enhanced as temperature increased.Z. ramigera beads were stable during all treatments and for prolonged periods of time (21 days).

Cadmium sorption by bacteria and freshwater sediment

SummarySorption of cadmium by sediment bacteria and freshwater sediment was investigated using diffusion chambers to simulate the water-sediment interface. Diffusion chambers were constructed to provide two compartments separated by a dialysis membrane. Diffusion of cadmium across the membrane was monitored after pure cultures of sediment bacteria or lake sediments were added to the sediment side of a diffusion chamber. Cellular accumulation of cadmium by cadmium-sensitive and cadmium-resistant bacteria removed between 20% and 80% of the dissolved cadmium from the simulated water column and pore water. Cellular accumulation of cadmium was greatest for cadmium-sensitive isolates that were tested. Sediment with an intact microbial community sequestered 80% of the cadmium added to sediment, whereas autoclaved sediment retained 97% of the metal that was added. Addition of glucose to cadmium-amended sediment decreased retention of cadmium by untreated and autoclaved sediments, resulting in elevated concentrations of dissolved cadmium in the simulated water column.