William F. Burke

NADP+-specific isocitrate dehydrogenase of Escherichia coli. II. Subunit structure.

Abstract The subunit structure and molecular weight of Escherichia coli NADP+-specific isocitrate dehydrogenase has been determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate at pH 7.0 and in 8 M urea at pH 2.9. The results obtained indicate that the enzyme, which exists as a catalytically active dimer, is composed of two subunits each with a molecular weight of approximately 42 000. Cross-linking with dimethylsuberimidate, followed by electrophoresis in sodium dodecylsulfate gives, in addition to a band of mol. wt 43 000, a second protein band of approximately 83 000. A single NH2-terminal amino acid, methionine, was obtained following dansylation of the performic acid oxidized protein suggesting identity of the subunits. The isoelectric point of the E. coli isocitrate dehydrogenase was found to be pH 5.0.

Crystallization of NADP-Specific Isocitrate Dehydrogenase

The first crystalline preparation of isocitrate dehydrogenase specific for nicotinamide adenine dinucleotide phosphate has been obtained with enzyme isolated from Escherichia coli. Scanning electron microscopy was employed to elucidate the structure of the crystals, which were found to exist as regular octahedrons ranging in size from 5 to 90 micrometers.

Genetics of Bacillus sphaericus

Since it was first described by Neide in 1904, Bacillus sphaericus has been the subject of a number of studies to refine the taxonomy of this species. However, for the most part the species still remains an enigma. Early taxonomic investigations placed considerable emphasis on spore morphology and position within the sporangium and on nutritional properties of the vegetative cells. However, because isolates assigned to this species are generally unreactive toward the substrates routinely used in taxonomic studies of Bacillus (Gordon, Haynes, and Pana 1973),B. sphaericus is considered somewhat of a catchall species. This conclusion has been supported by a variety of genetic and biochemical techniques.

Stability of pUB110 and pUB110-derived plasmids in Bacillus sphaericus 2362

The segregational and structural stability of pUB110 and four derivatives of various insert size and location was examined in the mosquito pathogen Bacillus sphaericus 2362 grown under conditions relevant to use of the bacterium as a larval control agent. Plasmids pUB110 (4.5 kb), pLDT103 (7.6 kb), and pTST130 (6.5 kb) exhibited 95–100% segregational stability at growth temperatures of 28° C and 38° C and at generation times of 42 and 108 min during 40 generations in a chemostat. Plasmids pLDT117 (9.7 kb) and pTST112 (6.5 kb), which had deletions in the BA4 membrane-binding site of the plasmids, were almost as stable (92–99%) unter these conditions. However, under growth conditions that allowed sporulation in mosquito larval cadavers, in batch culture or in the chemostat, plasmids with BA4-region deletions exhibited only 77–88% (pLDT117) or 66–81% stability in the resulting spores. Whereas segregational instability was associated with interruption of the BA4 region of these plasmids, this instability was primarily associated with the sporulation phase of development rather than with vegetative growth. Structural instability was not detected.

Molecular weight of Escherichia coli NADP-isocitrate dehydrogenase

The molecular weight of NADP-specific isocitrate dehydrogenase from Escherichia coli is unaffected by protein concentration, pH from 4.0 to 8.3, ionic strength over a 100-fold concentration range or by the presence of absence of substrates or co-factors.

Ultraviolet sensitivity ofBacillus subtilis citD mutants.

Cells ofBacillus subtilis168 with deletions in thecitD locus were found to be sensitive to irradiation with ultraviolet light and to mitomycin C but were able to repair DNA damage induced by methyl methanesulfonate. The recombination abilities of these cells, as determined by transformation and PBS1-mediated transduction experiments, were unaffected by the deletion. These phenotypic characteristics do not result from a metabolic imbalance caused by the deficiency of a functional α-ketoglutarate dehydrogenase complex, but most likely are a consequence of a genetic locus involved in ultraviolet repair, which is deleted together with thecitK gene when the deletion is formed.

Modification of the drop dialysis technique for use with larger volume samples

Desalting of nucleic acids by the drop dialysis method is limited by the fact that only small volume samples can be used due to the lack of sample containment on the membrane filters. A specially modified Styrafoam cup can be used as a membrane filter holder which serves to contain the sample, thus permitting dialysis of larger sample volumes.