Tatsuaki Tokuyama

Pure isolation of a new chemoautotrophic ammonia-oxidizing bacterium on gellan gum plate

Abstract A chemoautotrophic ammonia-oxidizing bacterium (strain TK794) was screened from eighty-eight soil samples. One bacterium was purely isolated on a Gellan gum-solidified plate. The cell of strain TK794 was 0.5–1.0 × 1.5–2.0 μm in size, rod-shaped, Gram-negative, obligately aerobic and motile by means of subpolar fragella. Intracytoplasmic membranes characteristic of nitrifying bacteria were present. The G+C-content of the DNA was 50.6 mol%. The optimum concentrations of phosphate and ammonium sulfate for maximum cell yield in P-medium were 70 mM and 30 mM, respectively. The c-type cytochromes were thought to be of the c-552 type. Growth of the strain was possible in the presence of cephalothin sodium (500 μg/ml). Strain TK794 was identified as belonging to the genus Nitrosomonas, but had enzymologically different characteristics from Nitrosomonas europaea ATCC 25978, the type strain.

Succession and community composition of ammonia-oxidizing archaea and bacteria in bulk soil of a Japanese paddy field

Abstract The present study describes succession in the abundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the upland and flooded bulk soils of a Japanese rice paddy field over 2 years using a quantitative polymerase chain reaction of both crenarchaeotal and betaproteobacterial ammonia monooxygenase alpha subunit (amoA) genes. A marked increase in the abundance of AOA amoA gene was observed in upland bulk soil after plowing, drainage and rice harvesting. A marked increase was also observed in the abundance of AOB amoA gene after plowing. The abundances of both AOA and AOB amoA genes in flooded bulk soil decreased immediately after flooding. During the middle period of flooding, the abundance of AOA amoA gene increased slightly in the flooded bulk soil. As the flooding subsided, the abundance of AOA amoA gene decreased, whereas that of AOB amoA gene increased. The AOA amoA gene sequences were affiliated with two phylogenetic clusters previously found in marine and soil environments. Both Nitrosospira-like and Nitrosomonas-like clones were detected. Our results revealed that there was a difference in the succession of abundances between AOA and AOB amoA genes in Japanese bulk paddy soil.

Characteristics of a high-concentration-ammonium sulfate-requiring ammonia-oxidizing bacterium isolated from deodorization plants of chicken farms.

A high-concentration-ammonium sulfate-requiring, ammonia-oxidizing bacterium, strain K1, was newly isolated from packed tower biological deodorization plants of chicken farms. The cells of strain K1 are rods (0.1-1.0 x 1.0-2.0 microm), gram negative, obligately aerobic, and nonmotile. Colonies (1-2 mm in diameter) on a plate culture are reddish, circular, and smooth. Intracytoplasmic membranes characteristic of nitrifying bacteria are present. The G+C content of the total DNA is 48.5 mol%. The similarity of 16S rRNA (%) to N. europaea ATCC 25978T (type strain) is 93.77%. This bacterium has a higher optimal growth temperature (35 degrees C) than is usually the case and tolerance up to 40 degrees C. The optimum concentration of ammonium sulfate in the medium is 303 mM, which should make it applicable for use in deodorization plants for enhancing the efficiency of deodorization. Phosphoglycerate kinase (PGK) and triosephosphate isomerase (TIM) were found to possess high specific activities (5700 and 4 x 10(5) U/mg, respectively) compared to the activities of these enzymes in strain ATCC 25978T (300 and 14 U/mg).

Nitrosomonas communis strain YNSRA, an ammonia-oxidizing bacterium, isolated from the reed rhizoplane in an aquaponics plant

An ammonia-oxidizing bacterium (strain YNSRA) was isolated from the rhizoplane of the reed (Phragmites communis) used in an aquaponics plant which is a wastewater treatment plant. Strain YNSRA was identified as Nitrosomonas communis by taxonomic studies. The hydroxylamine-cytochrome c reductase (HCR) of strain YNSRA was found to have a higher activity (25.60 u/mg) than that of Nitrosomonas europaea ATCC25978T (8.94 u/mg). Ribulose-1,5-bisphosphate carboxylase (RubisCO) activity was detected at very low levels in strain YNSRA, whereas strain ATCC25978T had definite activity.

A New Psychrotrophic Ammonia-Oxidizing Bacterium, Nitrosovibrio sp. TYM9

Abstract A new psychrotrophic ammonia-oxidizing bacterium (strain TYM9) was isolated in a screening of 150 soil samples on a gellan gum-solidified plate. The cells of strain TYM9 are slender cuved rods (0.2–0.4×1.0–3.0 μm), gram negative, obligately aerobic and nonmotile. Colonies (1–2 mm in diameter) on a plate culture are reddish, circular and smooth. Intracytoplasmic membranes characteristic of nitriying bacteria are absent. The G + C content of the DNA is 53.0 mol%. The optimum temperature for maximum growth in HEPES medium is 20°C and minimum growth occurs at 5°C and 35°C. The optimum concentration of ammonium sulfate for growth is 20 mM. Growth of the strain is possible in the presence of cephaloridine (500 μg/ml). Hydroxylamine oxidase (HAO) was detected with high specific activity (346 U/mg) compared to that for N. europaea ATCC 25978T (4.5 U/mg). The level of RuBisCO activity in strain TYM9 is similar to that in strain ATCC 25978T. Strain TYM9 has morphologically and physiologically different characteristics from those of Nitrosovibrio tenuis and members of the genus Nitrosomonas. Strain TYM9 was identified as belonging to the genus Nitrosovibrio, and assigned the Nitrosovibrio sp. TYM9.

Ribulose-1,5-bisphosphate carboxylase/oxygenase from an ammonia-oxidizing bacterium, Nitrosomonas sp. K1: purification and properties.

The ammonia-oxidizing chemoautotrophic Nitrosomonas sp. strain K1 exhibited marked ribulose-1,5-bisphosphate carboxylase (RubisCO) activity. The RubisCO [EC 4.1.1.39] was purified as an electrophoretically homogeneous protein. The molecular mass of the enzyme was estimated to be about 460 kDa by gel filtration, and it consists of two subunits [large (L): 52.2 kDa; small (S): 13.3 kDa] as demonstrated by SDS-PAGE. This confirmed that the enzyme has an L(8)S(8) structure. The K(m) values of the enzyme for RuBP, NaHCO3, and Mg2+ were estimated to be 0.112, 0.415, and 1.063 mM, respectively. The optimum pH and temperature for its activity were approximately 7.0 and 45 degrees C. The enzyme was stable up to 45 degrees C and in a pH range from 7.0-9.0 (4 degrees C, 48 h). The enzyme activity was inhibited by Cu2+, Hg2+, N-ethylmaleimide, p-chloromercuribenzoate, and SDS (0.1 mM). The activity was also inhibited by ammonium sulfate at high concentrations (38-303 mM) but the stability of the enzyme showed no inhibition at the same ammonium sulfate concentrations. The N-terminal amino acid sequences of the large and small subunits are AIKTYQAGVKEYRQTYW QPDYVPL and AIQAYHLTKKYETFSYLPQM, respectively.

Two kinds of ammonia-oxidizing bacteria isolated from biologically deodorizing plants in cold district.

Two kinds of ammonia-oxidizing bacteria isolated from biologically deodorizing plants in cold districts in Japan were identified as Nitrosomonas sp. IWT202 and Nitrosomonas sp. IWT514. The optimum pHs for growth were 8.0 (IWT202) and 7.5 (IWT514). Although rockwool samples for isolation were collected from the same plants, the optimum temperature for growth of strain IWT202 (37 degrees C) differed from that of strain IWT514 (30 degrees C). The bacteria had a higher (IWT202, 37 degrees C) and lower (IWT514, 20 degrees C) growth temperature than is usually the case. Both strains were shown to differ completely in regard to the effect of the ammonium sulfate concentration in the medium for a 20 degrees C culture and 30 degrees C culture. The inoculation of these bacteria provides the possibility of recovering ammonia-oxidizing activity, when the ammonia-oxidizing activity is lowered in biological deodorizing plants in cold districts. It seems that these strains are suitable for application to deodorization.

Newly isolated marine ammonia-oxidizing bacterium, Nitrosomonas sp. TNO632

Abstract A new marine ammonia-oxidizing bacterium (strain TNO632) was isolated in a screening of 80 sea-water samples on gellan gum-solidified plates. The cells of strain TNO632 are rods (0.7 – 0.9 × 1.0 – 2.0 μm), gramnegative, obligately aerobic, and nonmotile. Colonies (1–2 mm in diameter) on a plate culture are reddish, circular, and smooth. Intracytoplasmic membranes characteristic of nitrifying bacteria are present. The G+C content of the total DNA is 53.3 mol%. The similarity of 16S rRNA (%) to Nitrosomonas europaea ATCC 25978% (type strain) is 94.26%. The optimum temperature for maximum growth in marine ASW-medium is 27–29°C. The optimum concentrations of ammonium sulfate, sodium chloride, and magnesium chloride for growth are 26, 100 and 100 mM, respectively. Growth of the strain is imposiible when urea is used as the sole carbon source. Hydroxylamine cytochrome c reductase (HCR) and triosephosphate isomerase (TIM) were detected and found to possess high specific activities (980 and 140 U/mg, respectively) in TNO632 compared to those in ATCC 25978T (450 and 14 U/mg).

Characteristics of newly isolated ammonia-oxidizing bacteria from acid sulfate soil and the rhizoplane of leucaena grown in that soil

Abstract We isolated four new ammonia-oxidizing bacterial strains from leucaena (Leucaena leucocephala) grown in fields of an acid sulfate soil (ASS) in Thailand; 17SS from the surface soil and 17RS from the rhizoplane of a non-limed plot; 9SS from the surface soil and 9RS from the rhizoplane in a limed plot. The cells of all strains had the typical lobate shape of the genus Nitrosospira (“Nitrosolobus”). The percentage similarity of the 16S rRNA genes of these strains to that of Nitrosospira (“Nitrosolobus”) multiformis ATCC25196T (ATCC25196T) was 99.52% (strains 17SS, 17RS and 9SS) and 99.66% (strain 9RS). These newly isolated bacteria were all identified as Nitrosospira sp. Each strain, especially 17SS isolated from ASS, had higher urease activity than that of ATCC25196T. The o-acetylserine(thiol)lyase activity of 17SS was the highest of all the isolates. At pH 6.0, every isolate and ATCC25196T were able to utilize urea as the sole nitrogen source, in particular, strain 17SS grew best. The isolates from ASS showed higher urea utility than the isolates from the rhizoplane. Strain 17SS tolerated copper at levels up to 6.3 mmol L−1, but ATCC25196 T was inhibited at that concentration.

Purification and Some Properties of Citrate Synthase from a Nitrite-Oxidizing Chemoautotroph, Nitrobacter agilis ATCC 14123

Abstract Citrate (si)-synthase (citrate oxaloacetate-lyase, EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from a nitrite-oxidizing chemoautotrophic bacterium, Nitrobacter agilis ATCC 14123. The molecular mass (Mr) of the native enzyme was estimated to be about 250,000 by gel filtration, whereas SDS-PAGE gave two bands with Mr values of 45,000 and 80,000, respectively, suggesting that the enzyme is a tetramer consisting of two different subunits (α: 45,000, β: 80,000). The isoelectric point of the enzyme was 5.4. The pH and temperature optima on the citrate synthase activity were about 7.5–8.0 and 30–35°C, respectively. The citrate synthase was stable in the pH range of 6.0–9.0 and up to 55°C. The apparent Km values for oxaloacetate and acetyl-CoA were about 27 μM and 410 μM, respectively. The activity of citrate synthase was not inhibited by ATP (1 mM), NADH (1 mM) or 2-oxoglutarate (10 mM), but was strongly inhibited by SDS (1 mM). Activation by metal ions was not observed.