W. G. Martin

Differential effects of near-UV and visible light on active transport and other membrane processes in Escherichia coli.

Abstract— The effects of monochromatic near‐UV and visible light on active transport and several other membrane processes in Escherichia coli were investigated. Using mercury lines at 366, 405, 435, 546 and 578 nm, large differential effects were observed. Transport systems with photosensitive initial rates of uptake were classified into three groups on the basis Of wavelength dependence. Three, and possibly four photosensitizers may be involved; three active under aerobic conditions and the fourth in the absence of oxygen. Respiration rate exhibited the same sensitivity as one of the groups, suggesting that the active uptake of member amino acids (e.g. glycine) is largely dependent on oxidative energy. The photosensitivity of glycine transport at 405 nm was not the result of inhibition of the membrane‐bound Ca–Mg adenosine triphosphatase as shown using an isogenic mutant strain. Cell viability was not affected at the highly active wavelength, 405 nm. Photoeffects on transport of α‐methylglucoside were minimal at 366 and 405 nm, contrasting to most of the amino acids investigated. The relative photosensitivity of respiration and several amino acid transport systems depended on carbon source.

The identification and partial characterization of a plasmid containing the gene for the membrane-associated hydrogenase from E. coli

Escherichia coli DNA was digested with restriction endonuclease PstI and ligated into the PstI site of plasmid pBR322. Recombinant plasmids that were constructed in this manner were used to transform E. coli H61, a mutant with a decreased level of hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for the membrane-associated E. coli hydrogenase in plasmid pBL101. In E. coli minicells, the pBL101 DNA directed the synthesis of a protein of a size corresponding to that of the precursor of the E. coli membrane-associated hydrogenase, which appears to contain an uncleaved leader peptide. A restriction map of the cloned DNA was determined for 14 endonucleases.

PROTECTION FROM VISIBLE LIGHT DAMAGE TO ENZYMES AND TRANSPORT IN ESCHERICHIA COLI

Abstract— Cysteine, anthranilic acid and adenine, in decreasing order of effectiveness, protected flavo‐enzymes of E. coli against inactivation with high‐intensity, heterochromatic light above 400 nm. Cell suspensions illuminated for 40 min in the presence of cysteine retained 30, 40 and 55% of their succinate, l‐α‐glycerophosphate and d‐lactate dehydrogenase activities, whereas less than 10% activity could be detected in the absence of this protective agent. Different effects on respiration and transport systems were obtained with these protective agents. Adenine and anthranilic acid provided substantial protection of the glycine and methylthio‐ß‐d‐galactoside transport systems but failed to attenuate adverse light effects on respiration and phenylalanine uptake. Cysteine did not protect respiration and potentiated damage to selected transport systems. Our results suggest that flavins act as photosensitizers.

Bacillus thuringiensis parasporal crystal toxin: Dissociation into toxic low molecular weight peptides

Parasporal crystals of Bacillus thuringiensis can be dissociated into low molecular weight peptides (< 5000 daltons) by dissolving them in 0.1 M N-morpholinopropane sulfonic acid buffer pH 7.8 containing 0.05 M dithiothreitol and 2M–4M KSCN, or by performic acid oxidation. The peptides obtained by dissolving in KSCN were still toxic to silkworm larvae.

Application of Hydrophobic Chromatography to the Large Scale Purification of Pesulfovibri0 Desulfur Icans Cytochrome C3

Hydrophobic chromatography on Phenyl-Sepharose was employed for large scale purification of cytochrome c3 from Desulfovibrio desulfuricans. This chromatographic procedure minimizes operational volumes and eliminates the lengthy dialysis ordinarily used to remove large amounts of salts. Pure cytochrome c3 was obtained after one additional purification by ion-exchange chromatography.

Immobilization of isolated and cellular hydrogenase of D. desulfuricans in radiation-polymerized polyacrylamides

Purified hydrogenase fromDesulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon immobilization in contrast to 6.0 for the free enzyme and the range of 6–7 for whole cells. Both the purified enzyme and whole cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65‡C) and long term storage (4‡C) of the hydrogenase activity of both the purified enzyme and whole cells.

A relation between amino acid hydrophobicity and rate of uptake in Escherichia coli.

Abstract Fumarate and ferricyanide increased the rate of amino acid uptake in anoxic cells of E. coli suspended in a glycerol medium. The stimulation correlated with the hydrophobicity of 16 amino acids transported by several carrier systems. Fumarate and ferricyanide increased membrane energization as measured by changes in transmembrane pH and electrical potential, or by quenching of fluorescence of 1-anilino-8-naphthalenesulfonate. The results suggest that a common, rate determining step among the amino acid transport systems is the transfer of the substrate from an aqueous to an apolar environment.

Immobilization of Desulphovibrio desulphuricans: cell-associated hydrogenase in beaded matrices

Abstract The immobilization of the hydrogenase (cytochrome c 3 hydrogenase, hydrogen: ferricytochrome c 3 oxidoreductase, EC 1.12.2.1) activity associated with Desulphovibrio desulphuricans whole cells is described. The periplasmic hydrogenase was prevented from leaking from the cells by glutaraldehyde treatment. This modification left the hydrogenase activity of the cells unchanged. The resulting whole-cell preparation was then immobilized in beaded gels formed by either calcium alginate or low temperature radiation-polymerized polyacrylamide(s). The alginate matrix was used to study the effect of bead diameter and retention of hydrogenase activity after immobilization. Polyacrylamide matrices were used to study the effects of immobilization on hydrogenase activity vs. pH profiles and also matrix charge effects on enzymatic activity and long-term storage.