Hohzoh Kiyohara

A locus of Pseudomonas pickettii DTP0602, had, that encodes 2,4,6-trichlorophenol-4-dechlorinase with hydroxylase activity, and hydroxylation of various chlorophenols by the enzyme

Abstract Pseudomonas pickettii DTP0602 utilizes 2,4,6-trichlorophenol (2,4,6-TCP) as a sole source of carbon and energy. 2,4,6-TCP is dechlorinated and converted to 2,6-dichlorohydroquinone by a primarily attacking enzyme of catabolism. The genes encoding the enzyme were cloned using a transposon tagging strategy in Escherichia coli and Pseudomonas putida . A kanamycin-resistant strain derived from P. pickettii DTP0602 by Tn5 insertion, which was named DTP6251, had less dechlorinase activity of 2,4,6-TCP than the parent strain, and 2,6-dihydroquinone accumulated in the culture broth of this mutant. A DNA fragement containing Tn5 together with its flanking region was isolated from strain DTP6251. Deletion and subcloning analysis of the fragment showed that a 3.5-kb region flanked by Tn5 was essential for dechlorinase activity. Two open reading frames denoted hadA and hadB were located in the region: hadA spanned 1,554 nucleotides and encoded a polypeptide with a deduced molecular mass of 58,540; hadB spanned 591 nucleotides and encoded a polypeptide with a deduced molecular mass of 21,167. A set of promoter sequences ( σ 54 recognition sequence; -GG-…-GC- at positions −24 and −12) was found upstream of hadA . Two polypeptides were produced when this region was expressed under the control of the tac and trc promoters in E. coli . HadA was a chlorophenol-4-hydroxylase that hydroxylated various chlorophenols other than 2,4,6-TCP at position 4 to yield corresponding p -dihydroquinones. HadA seemed to be a flavoprotein because FAD and NADH were required for its hydroxylation activity in vitro . Even though both hadA and hadB were essential for expression of hydroxylase activity in vivo , the mixture consisting of HadA and NADH (and/or FAD) expressed hydroxylase activity in vitro . The product of the hadB gene ( i.e. , HadB) was not essential for expression of dechlorinase activity in vitro .

Natural distribution of bacteria metabolizing many kinds of polycyclic aromatic hydrocarbons

Abstract Bacteria that can metabolize at least six polycyclic aromatic hydrocarbons (biphenyl, naphthalene, phenanthrene, anthracene, pyrene, and chrysene) were found to be distributed widely in nature. The distribution of bacteria that could metabolize phenanthrene was independent of petroleum pollution. All isolates of bacteria metabolizing pyrene or anthracene could also metabolize phenanthrene.

Cloning and sequence analysis of hydroxyquinol 1,2-dioxygenase gene in 2,4,6-trichlorophenol-degrading Ralstonia pickettii DTP0602 and characterization of its product.

A gene encoding hydroxyquinol 1,2-dioxygenase was cloned from 2,4,6-trichlorophenol-degrading Ralstonia (Pseudomonas) pickettii strain DTP0602. Cell-free extracts of Escherichia coli containing a cloned 1.4-kb StuI-XhoI DNA fragment of R. pickettii DTP0602 hydroxyquinol 1,2-dioxygenase converted hydroxyquinol into maleylacetate and also degraded 6-chlorohydroxyquinol. The 1.4-kb DNA fragment contained one open reading frame (designated hadC) composed of 948 nucleotides. The molecular mass of 34,591 deduced from the gene product (HadC) was in agreement with the size (35 kDa) of the purified HadC protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of HadC exhibited high homology to that of the hydroxyquinol 1,2-dioxygenase of 2,4,5-trichlorophenoxyacetic acid-degrading Burkholderia cepacia AC1100 (Daubaras, D. L. et al., Appl. Environ. Microbiol., 61, 1279-1289, 1995). The active enzyme had a molecular mass of 68 kDa, suggesting that it is functional as a homodimer. The enzyme also catalyzed the oxidation of pyrogallol and 3-methylcatechol, possible intermediates in the degradation of 2,4,6-trichlorophenol, in addition to 6-chlorohydroxyquinol and hydroxyquinol. The dioxygenase catalyzed both ortho- and meta-cleavage of 3-methylcatechol.

Meta-fission product hydrolases from a strong PCB degrader Rhodococcus sp. RHA1

Abstract Both 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) and 2-hydroxy-6-oxohepta-2,4-dienoic acid (HOHD) hydrolases were purified to homogeneity from the cells of PCB-degrader, Rhodococcus sp. RHA1 grown on biphenyl. The NH 2 -terminal amino acid sequences of these hydrolases were determined and low homology (35%) was observed between them. Both of them were induced in the RHA1 cells grown on benzene, toluene and ethylbenzene as well as on biphenyl, but not induced in those grown on benzoate, phenol and succinate. The RHA1 HPDA and HOHD hydrolases were indicated to be an octamer and a dimer respectively composed of identical subunits with a molecular weight of 33,000 and 32,000, respectively. The RHA1 HPDA and HOHD hydrolases were specific for the respective substrates HPDA and HOHD, and the latter also had high activity toward 2-hydroxy muconic semialdehyde (HMSA). Such narrow substrate specificity suggested that HPDA and HOHD hydrolases were responsible for the degradation of biphenyl and toluene, respectively.

Nucleotide sequences and characterization of genes encoding naphthalene upper pathway of pseudomonas aeruginosa PaK1 and Pseudomonas putida OUS82.

A 12,808-nucleotide containing DNA fragment cloned from naphthalene-utilizing (Nah+) Pseudomonas aeruginosa PaK1 was analyzed and compared with the genes (pah(OUS)) of a 14,462-nucleotide DNA fragment from Pseudomonas putida OUS82. The DNA sequence analyses demonstrated that the naphthalene upper-pathway genes and their deduced enzymes were very similar between the two bacteria: nucleotide similarities, 83-93%; amino acid similarities, 79-95%. These genes were also similar to those of the nah operon of plasmid NAH7; in particular, the OUS82 genes were similar to the nah genes, whereas the PaK1 genes were almost identical to the dox genes of Pseudomonas sp. C18. A region homologous with the 84-bp repeated sequence that Eaton (J. Bacteriol., 176, 7757-7762, 1994) has found at a site upstream of he nah operon was found only in a region downstream of the pah(PaK) gene cluster in PaK1 and on both sides of the pah(OUS) gene cluster in OUS82. A PaK1 gene, corresponding to an unknown gene (nahQ) in the nah operon, is located between the 1,2-dihydroxynaphthalene dioxygenase gene and the trans-o-hydroxybenzylindenepyruvate (tHBP A) hydratase-aldolase gene (nahE), and was suggested to be involved in the conversion of naphthalene to salicylate. Just downstream of the pah(PaK) gene cluster, a portion of a region was identical to one-third of the transposase gene (tnpA) in a phenol-catabolic plasmid pEST1226.

Plasmid involvement in the degradation of polycyclic aromatic hydrocarbons by a Beijerinckia species.

A Beijerinckia species, capable of oxidizing phenanthrene, biphenyl and other polycyclic aromatic hydrocarbons, was shown to contain two plasmids that were designated pKGl and pKG2. The molecular masses of plasmids pKG1 and pKG2, as determined by electron microscopy, were approximately 147 X 10(6) and 20.8 X 10(6) daltons, respectively. Growth of the organism on benzoate led to the isolation of strains that had lost the ability to grow with phenanthrene and biphenyl. All of the Phn-, Bph- strains had also lost the smaller plasmid, pKG2. The results presented suggest that plasmid pKG2 is responsible for the synthesis of enzymes involved in the degradation of phenanthrene and biphenyl.

Characterization of a phenanthrene degradation plasmid from Alcaligenes faecalis AFK2.

Abstract A plasmid pHK2 involved in phenanthrene degradation was isolated from Alcaligenes faecalis AFK2. Some mitomycin C treated cells lost the pHK2 plasmid. The plasmid could be transformed into Pseudomonas putida and A. faecalis. They gained the ability to utilize phenanthrene as well as o-phthalate, an intermediate of phenanthrene degradation. Phenanthrene dioxygenase could be detected from the transformants after induction with phenanthrene. The molecular size of pHK2 DNA was determined to be 42.5 kilobase (kb), and its physical map was constructed.

Intergeneric hybridization betweenMonascus anka andAspergillus oryzae by protoplast fusion

SummaryTo breed industrially useful strains of a slow-growing, red-pigment-producing strain ofMonascus anka, protoplasts ofM. anka MAK1 (arg) andAspergillus oryzae AOK1 (met, thr) were fused. A mixture of protoplasts prepared from mycelia ofM. anka MAK1 treated with 2% Usukizyme and ofA. oryzae AOK1 treated with 2% Usukizyme and 0.2% NovoZym 234 was incubated with 30% (w/v) polyethylene glycol no. 6000. Heterokaryon fusants complementing the auxotrophies of both mutants were isolated on minimal medium, but segregated into red (MAK1) and white (AOK1) sectors after being cultured on a complete medium. After irradiation with UV light, the fusants gave stable heterozygous diploids that formed long white hyphae. These diploids, which had twice as much DNA in the nucleus as their parents, grew more rapidly than the parent strain YZT1, and produced ethanol earlier than the parents. Production of amylase, protease, and kojic acid by the fusants was intermediate in amount between that of the two parents.

Degradability of polychlorinated phenols by bacterial populations in soil

Abstract The biodegradabilities of polychlorinated phenols including 5 isomers of trichlorophenols, 3 isomers of tetrachlorophenols and pentachlorophenol, were tested with 170 samples of soil collected from various environments. After the samples were inoculated into a succinate-containing mineral medium and incubated, the cultures were acclimatized to phenol concentrations from 10 to 100 ppm. Twenty six samples (15%) were observed to degrade 2, 4, 6-trichlorophenol (246TrCP) and a mixed sample of soil degraded 2, 3, 4, 6-tetrachlorophenol, but no degradation was seen with other chlorophenols. All of the mixed cultures acclimatized to and degrading 246TrCP also degraded phenol. For the degradation of 246TrCP, the NO3′ ion was preferred to the NH4+ ion as a nitrogen source. At concentrations below 500 ppm, 246TrCP was degraded completely within 8 d and the chloride ion was detected in the culture broth at an amount corresponding to that of the chlorinated phenol, although cell growth was inhibited at a 246TrCP concentration of 1,000 ppm. No possible intermediate product of 246TrCP was detected in the cultures.

Restoration of the ability to settle bulking sludge by bacterial seeding in wastewater treatment

An attempt to restore the settling ability of denatured bulking sludge was under-taken by changing the microflora in waste treatment tanks. A cell suspension of mixed cultures of ten strains of bacteria, which were isolated from normal activated sludge from night soil plants, and a type culture Zoogloea ramigera IAM1236 was seeded into a laboratory-scale aeration tank containing bulking sludge collected from municipal night soil or a food processing (bean curd production) waste treatment plant. The tank was fed with synthetic wastewater or industrial waste and aerated for 22 days. After 5 days, the microflora in the sludge changed remarkably with the seeding of the bacterial culture; filamentous organisms disappeared and active protozoa (Vorticella sp., Epistylis sp., and Lecane sp.) appeared. The sludge became compact and settled rapidly. The SV30 of the sludge temporarily increased, but in the end decreased from 97 to 20%. The CODcr value decreased from 300 to 20 ppm. In the tank without seeding, the sludge contained almost filamentous organisms only which floated and finally decomposed. The effects were confirmed by applied tests in 700-ton scale and 100-ton scale aeration tanks of municipal night soil and a food processing waste treatment plant, respectively.