Use of 4-Nitrophenoxyacetic Acid for Detection and Quantification of 2,4-Dichlorophenoxyacetic Acid (2,4-D)/(alpha)-Ketoglutarate Dioxygenase Activity in 2,4-D-Degrading Microorganisms

Purified 2,4-dichlorophenoxyacetic acid (2,4-D)/(alpha)-ketoglutarate dioxygenase (TfdA) was shown to use 4-nitrophenoxyacetic acid (K(infm) = 0.89 (plusmn) 0.04 mM, k(infcat) [catalytic constant] = 540 (plusmn) 10 min(sup-1)), producing intensely yellow 4-nitrophenol. This reagent was used to develop a rapid, continuous, colorimetric assay for the detection of TfdA and analogous activities in 2,4-D-degrading bacterial cells and extracts.     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

Nitrobacter winogradskyi cytochrome a1c1 is an iron-sulfur molybdoenzyme having hemes a and c

Cytochrome a1c1 (nitrite-cytochrome c oxidoreductase) purified from Nitrobacter winogradskyi (formerly N. agilis) contained molybdenum, non-heme iron, and acid-labile sulfur in addition to hemes a and c; it contained 1 mol of heme a, 4-5 g atoms of non-heme iron, 2-5 g atoms of acid-labile sulfur, and 1-2 g atoms of molybdenum per mol of heme c, but did not contain copper. The fluorescence spectra of the molybdenum cofactor derivative prepared from cytochrome a1c1 were very similar to those of the cofactor derivative from xanthine oxidase, and the aponitrate reductase of nit-1 mutant of Neurospora crassa was complemented by addition of the molybdenum cofactor derived from the cytochrome. Further, the ESR spectrum of cytochrome a1c1 was similar to that of liver sulfite oxidase. The content of cytochrome a1 in the cells cultivated with the medium in which tungsten was substituted for molybdenum markedly decreased as compared with that in the cells cultivated in the molybdenum-supplemented medium. These results indicate that cytochrome a1c1 is an iron-sulfur molybdoenzyme which contains hemes a and c.     

Nucleotide sequences of two cellulase genes from alkalophilic Bacillus sp. strain N-4 and their strong homology.

Two genes for cellulases of alkalophilic Bacillus sp. strain N-4 (ATCC 21833) have been sequenced. From the DNA sequences the cellulases encoded in the plasmids pNK1 and pNK2 consist of 488 and 409 amino acids, respectively. The DNA and protein sequences of the pNK1-encoded cellulase are related to those of the pNK2-encoded cellulase. The pNK2-encoded cellulase lacks the direct repeat sequence of a stretch of 60 amino acids near the C-terminal end of the pNK1-encoded cellulase. The duplication of the cellulase genes and the formation of the direct repeat in the pNK1-encoded cellulase occurred at almost the same time.     

Gene responsible for dificient activity of the β subunit of C8, the eighth component of complement,is located on mouse chromosome 4

Sera from 73 strains of mice were tested for hemolytic activity through the classical and the alternative pathways (CP and AP) in a single radical hemolysis assay. Sera from 16 out of 45 laboratory inbred strains had n o lytic activity, and Ouchternoly analysis with anti-C5 serum showed them to be C5-deficient. Sera from 2 out of 28 strains derived from wild mice also had no lytic activity, but the C5 molecule was detectable in both. The hemolytic activity of sera from these strains can be restored serum deficient in C8-, leading us to conclude that strains M.MOL-MSM (MSM) and Mae are deficient in the subunit of C8. Typing of (DBA/2J ×MSM)F₁ hybrids and of progeny of a backcross to MSM showed that this C8 deficiency is controlled by a singles recessive gene, designated C8b; the allele with hemolytic activity is C8b ¹; and the allele with no activity C8b ⁰. Because of synteny homologies in mouse and human , we looked for and found close linkage between C8b and Pgm-2. Typing of recombinant mice for Mup-1 mapped the C8b locus 2.3 centimorgans (cM) telomeric to Pgm-2 on mouse chromosome 4.     

Actual distribution of bacteriocytes in the trophosome of a beard worm (Oligobrachia mashikoi, Siboglinidae, Annelida): clarification using whole-mount in situ hybridization

Beard worms (Siboglinidae, Polychaeta) lack a mouth and a digestive tract and harbour chaemosynthetic bacteria in the bacteriocytes of the trophosome. Since beard worms depend on the organic compounds produced by the bacteria for nourishment, the bacteriocytes should be efficient in exchanging various substances with body fluids. For this reason, it is important to determine how the bacteriocytes are organized in the trophosome. As the first step of the present study, the appearance of bacteriocytes was examined in routinely stained paraffin sections. Second, visualization of the actual distribution of the bacteriocytes was attempted using whole‐mount in situ hybridization with a probe of the 16S rRNA nucleotide sequence of the bacterium. After routine haematoxylin & eosin staining, the bacteriocytes appeared to be aligned in cell cords accompanied with nutrient‐deposit cells that extended from both sides of the trophosome toward the dorsal side and folded up in the coelomic spaces. In whole‐mount preparations, however, bacteriocytes with intense signals of 16S rRNA were seen three‐dimensionally as many irregular leaves arranged from both sides of the ventral vessel toward the dorsal vessel. We will discuss the physiological significance of this characteristic distribution of the bacteriocytes in the present species.     

Reactivity of the co-type and baa 3-type cytochrome c oxidases from Pseudomonas aeruginosa with different endogenous cytochromes c

The reactivity between different cytochromes c purified from Pseudomonas aeruginosa cells grown aerobically in the absence of nitrate and isolated cytochromes co and baa ₃ was determined. The P. aeruginosa cytochrome co reacted most rapidly with the membrane-bound cytochrome c-551 among three c-type cytochromes analyzed, whereas the cytochrome baa ₃ reacted best with the membrane-bound cytochrome c-555. The results indicated that two terminal electron transfer systems are present in aerobic P. aeruginosa: one contains the cytochrome c-551 and cytochrome co, and the other contains the cytochrome c-555 and cytochrome baa ₃.     

Tandem location of the cellulase genes on the chromosome of Bacillus sp. strain N-4

Abstract A 5.7-kb Eco RI DNA fragment has been isolated from Bacillus sp. strain N-4 chromosome DNA. This fragment contained both the pNK1-encoded cellulase ( celB ) gene and the pNK2-encoded cellulase ( celA ) gene which were highly homologous [13]. These results demonstrate the tandem location of these genes on the chromosomal DNA. The homologous sequence, which may play an important role for the gene duplication, were observed 5' upstream of the celA gene, between the celA and celB genes, and 3' downstream from the celB gene.