A high-molecular-weight c-type cytochrome, Cyc2, and a putative 22-kDa c-type cytochrome were detected in the membrane fraction released during spheroplast formation from Acidithiobacillus ferrooxidans. This fraction was enriched in outer membrane components and devoid of cytoplasmic membrane markers. The genetics, as well as the subcellular localization of Cyc2 at the outer membrane level, therefore make it a prime candidate for the initial electron acceptor in the respiratory pathway between ferrous iron and oxygen.

The nitrate reductase of Escherichia coil K-12 was localized in a particulate fraction of the cell and it sedimented as if it were bound to a large substructure that is subject to fragmentation during cell disruption procedures. Soluble enzyme, exhibiting a homogenous profile in sucrose gradients, was released from this fraction by an alkaline-heat treatment. Less than 1.5% of total active nitrate reductase apparently occurred in this soluble form during the course of formation of the particulate enzyme. Enzyme synthesis was repressed by aeration in the presence or absence of nitrate. Under anaerobic conditions, nitrate reductase was synthesized at a rate that could be increased 20-fold by the addition of nitrate. When enzyme synthesis was initiated by induction with nitrate or anaerobiosis, biphasic kinetics were obtained. We interpreted the results as evidence for the existence of a redoxsensitive repressor which mediates nitrate reductase regulation. The complex of respiratory enzymes found in E. coli grown aerobically with (2) and without EKscherichia coli K-12 appears to be an ideal sub-(16) nitrate and anaerobically on both complex ject for the study of the synthesis and regulation (15) and synthetic media (1, 5). Various factors of organized elements in cells. Subject to elaboseem to affect the synthesis and regulation of the rate control mechanisms, the synthesis of many enzyme. Because the varied and sometimes conof these enzymes can be initiated or repressed by flicting findings on the nature of nitrate reductase varying the culture conditions. Because these en-and the effects of environmental factors on its zymes are nonobligatory for the growth of a synthesis have been obtained with different facultative anaerobe, the isolation and study of strains of E. coli, we undertook a study of the mutants are facilitated. We initiated such a study physical state and mode of regulation of the with the investigation of nitrate reductase. This is enzyme in a single strain, E. coli K-12. the last enzyme in an electron-transport chain which enables E. coli to utilize nitrate instead of MATERIALS AND MoDS oxygen as a terminal electron acceptor (11, 12), Bacterial strains. Experiments on the effects of carrying out what has been termed "anaerobic various cell disruption procedures were performed respiration." with E. coll K-10 obtained from M. Santer. Otherwise, Nitrate reductase has been studied in several the following two strains of E. coli K-12 were utilized different higher plants and bacteria, including-train 3300 which is constitutive forfl-galactosidase, some strains of E. coli. These studies have been and strain 3000, which is inducible for this enzyme. reviewed by Tamguchi (14) and by Nason (7). ResultrKbtained in strain K-10 were confirmed in The functional properties and physical charac-Media. All liquid media employed the minimal teristics of the enzyme found in different organ-salts base of Sypherd and Strauss (13), supplemented isms vary widely. Nitrate reductase in E. coli with 50 4g of thiamine HCI pe...

No abstract

The effects of adding molybdate and selenite to a glucose-minimal salts medium on the formation of enzymes involved in the anaerobic metabolism of formate and nitrate in Escherichia coli have been studied. When cells were grown anaerobically in the presence of nitrate, molybdate stimulated the formation of nitrate reductase and a b-type cytochrome, resulting in cells that had the capacity for active nitrate reduction in the absence of formate dehydrogenase. Under the same conditions, selenite in addition to molybdate was required for forming the enzyme system which permits formate to serve as an effective electron donor for nitrate reduction. When cells were grown anaerobically on a glucose-minimal salts medium without nitrate, active hydrogen production from formate as well as formate dehydrogenase activity depended on the presence of both selenite and molybdate. The effects of these metals on the formation of formate dehydrogenase was blocked by chloramphenicol, suggesting that protein synthesis is required for the increases observed. It is proposed that the same formate dehydrogenase is involved in nitrate reduction, hydrogen production, and in aerobic formate oxidation. 1006 on August 5, 2020 by guest http://jb.asm.org/ Downloaded from phate (pH 7.2), 0.25 mm sodium EDTA, and 1.0 mM 1007

No abstract