Deoxyribonucleic acid in Nitrobacter carboxysomes.

Carboxysomes were isolated from Nitrobacter winogradskyi and Nitrobacter agilis. The icosahedral particles contained double-stranded deoxyribonucleic acid (DNA). In the presence of ethidium bromide and cesium chloride, the particle-bound DNA had a buoyant density of rho 25 = 1.701 g/cm3. Electron microscopy revealed the DNA to be a 14-micron circular molecule.     

Stimulation of Nitrobacter agilis by Biotin.

Krulwich, Terry A. (Goucher College, Baltimore, Md.), and Helen B. Funk. Stimulation of Nitrobacter agilis by biotin. J. Bacteriol. 90:729-733. 1965.-Addition of biotin to nitrite-mineral medium greatly stimulated the autotrophic growth of four strains of Nitrobacter agilis. Comparisons of cultures of the organisms grown in parallel at 30 C in nitrite medium and in the medium supplemented with 150 mmug of biotin per ml showed that the vitamin promoted: (i) 2- to 4-fold greater rates of utilization of nitrite, and (ii) 100- to 1,000-fold greater populations of cells per milliliter. Avidin specifically inhibited the biotin stimulation of nitrite utilization at an avidin-biotin ratio of 133:1. Incubation of the four strains of N. agilis at 37 C imposed a requirement for biotin that could be met by daily addition of 150 mmug of the vitamin per ml of medium. The stimulatory effects of the vitamin at 30 C suggest that in N. agilis the synthesis of biotin is rate-limiting for growth.     

Lack of Distinction Between Nitrobacter agilis and Nitrobacter winogradskyi

No adequate criteria were established to distinguish between Nitrobacter agilis and N. winogradskyi. However, very gentle preparative techniques permitted demonstration of flagella in N. agilis.     

Permeability of Nitrobacter agilis to Organic Compounds.

Ida, S. (Cornell University, Ithaca, N.Y.), and M. Alexander. Permeability of Nitrobacter agilis to organic compounds. J. Bacteriol. 90:151-156. 1965.-None of a variety of inorganic ions or organic compounds served as a sole energy source for the growth of Nitrobacter agilis, and the test substrates were not oxidized by either intact cells or extracts of the obligate chemoautotroph. The organic substances did not serve as sole carbon sources for the bacterium in a synthetic medium, and they failed to enhance the rate of nitrite oxidation. The organism was permeable to acetate and a number of other simple carbon compounds, however, and exogenously supplied acetate was converted to a number of products. On the basis of these findings, possible reasons are examined for the inability of the chemoautotroph to use exogenous organic compounds as energy or carbon sources.     

Inhibitor Evaluation with Immobilized Nitrobacter agilis Cells

Nitrobacter agilis was entrapped in calcium alginate beads and used as a floating bed supplied with a continuous flow of nitrite medium. Complete nitrite oxidation was achieved within 30 h, and the system could be maintained for at least 210 h. The immobilized Nitrobacter system was subjected to sulfur oxyanions, acidity, and metal ions. Thiosulfate and tetrathionate (up to 20 mM each) did not inhibit the nitrite oxidation activity. A low pH of 4.2 resulted in the complete cessation of nitrite oxidation, and the activity was not restored upon increasing the pH to 7. Nitrite oxidation by N. agilis was sensitive to 10 mM each Ni²⁺ and Al³⁺ but insensitive to 10 mM MoO₄²⁻.     

Effects of Pesticides on Nitrite Oxidation by Nitrobacter agilis

The influence of pesticides on the growth of Nitrobacter agilis in aerated cultures and on the respiration of N. agilis cell suspensions and cell-free extracts was studied. Two pesticides, aldrin and simazine, were not inhibitory to growth of Nitrobacter, but five compounds [isopropyl N-(3-chlorophenyl) carbamate (CIPC), chlordane, 1,1-dichloro-2,2-bis (p-chlorophenyl) ethane (DDD), heptachlor, and lindane] prevented growth when added to the medium at a concentration of 10 mug/ml. Whereas CIPC and eptam prevented nitrite oxidation by cell suspensions, the addition of DDD and lindane resulted in only partial inhibition of the oxidation. Heptachlor and chlordane also caused only partial inhibition of oxidation, but were more toxic with cell-free extract nitrite oxidase. None of the pesticides inhibited the nitrate reductase activity of cell-free extracts, but most caused some repression of cytochrome c oxidase activity. Heptachlor was the most deleterious compound.     

Nitrite oxidase and nitrate reductase in Nitrobacter agilis

Nitrite oxidase and nitrate reductase in Nitrobacter agilis were shown to be separate enzymes. The best separation of the two systems was achieved by ammonium sulphate fractionation. The effects of various compounds, including antimycin A, 2-n-heptyl-4-hydroxyquinoline N-oxide and chlorate, also clearly distinguish between the two enzyme reactions. The relationship between the two opposing reactions in Nitrobacter is discussed.     

Ultrastructure of Nitrobacter agilis Grown Under Autotrophic and Heterotrophic Conditions

Nitrobacter agilis, grown through seven transfers heterotrophically in the absence of nitrite, was examined in the electron microscope. The ultrastructure of such cells closely resembled that of autotrophically grown N. agilis. It was thus futher established that the organisms growing heterotrophically were indeed N. agilis and, therefore, that N. agilis is a facultative autotroph. Acetate incorporation into poly-beta-hydroxybutyrate was confirmed cytologically.     

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.     

The complete amino acid sequence of Nitrobacter agilis cytochrome c-550

The amino acid sequence of cytochrome c-550 from the chemoautotroph, Nitrobacter agilis, was completed by using solid-phase sequencing and conventional procedures. The cytochrome was composed of 109 amino acid residues and its molecular weight was calculated to be 12375 including haem c. The cytochrome was homologous to eukaryotic cytochromes c and some photosynthetic bacterial cytochromes c2. In particular, its primary structure was very similar to that of Rhodopseudomonas viridis cytochrome c2. Some of its properties were compared with those of other cytochromes c on the basis of the primary structure.