Noboru Takizawa

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.

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.

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.

Novel Denitrifying Bacterium Ochrobactrum anthropi YD50.2 Tolerates High Levels of Reactive Nitrogen Oxides

ABSTRACT Most studies of bacterial denitrification have used nitrate (NO3−) as the first electron acceptor, whereas relatively less is understood about nitrite (NO2−) denitrification. We isolated novel bacteria that proliferated in the presence of high levels of NO2− (72 mM). Strain YD50.2, among several isolates, was taxonomically positioned within the α subclass of Proteobacteria and identified as Ochrobactrum anthropi YD50.2. This strain denitrified NO2−, as well as NO3−. The gene clusters for denitrification (nar, nir, nor, and nos) were cloned from O. anthropi YD50.2, in which the nir and nor operons were linked. We confirmed that nirK in the nir-nor operon produced a functional NO2− reductase containing copper that was involved in bacterial NO2− reduction. The strain denitrified up to 40 mM NO2− to dinitrogen under anaerobic conditions in which other denitrifiers or NO3− reducers such as Pseudomonas aeruginosa and Ralstonia eutropha and nitrate-respiring Escherichia coli neither proliferated nor reduced NO2−. Under nondenitrifying aerobic conditions, O. anthropi YD50.2 and its type strain ATCC 49188T proliferated even in the presence of higher levels of NO2− (100 mM), and both were considerably more resistant to acidic NO2− than were the other strains noted above. These results indicated that O. anthropi YD50.2 is a novel denitrifier that has evolved reactive nitrogen oxide tolerance mechanisms.

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.

Analysis of Two Gene Clusters Involved in 2,4,6-Trichlorophenol Degradation by Ralstonia pickettii DTP0602

Ralstonia pickettii DTP0602 utilizes 2,4,6-trichlorophenol (2,4,6-TCP) as sole source of carbon and energy. We have characterized hadABC which is involved in the degradation of 2,4,6-TCP. To identify the other genes involved in 2,4,6-TCP degradation, the DNA sequence around hadABC was determined. A regulatory gene, hadR, homologous to the LysR-type transcriptional regulator was located upstream of hadA, but no maleylacetate (MA) reductase gene was located near hadABC. An 8.4-kb DNA fragment containing a MA reductase gene, hadD, was cloned using a DNA probe designed from the N-terminal sequence of purified MA reductase. hadD was located upstream of an open reading frame, hadS, which codes for a homolog of the LysR-type transcriptional regulator. A hadS insertion mutant, DTP62S, constitutively expressed MA reductase when grown on aspartate in the absence of 2,4,6-TCP. MA reductase was repressed in DTP62S supplemented with hadS. HadR and HadS are proposed to be a positive and a negative regulator, respectively. A draft genome sequence analysis revealed that the hadRXABC and hadSYD clusters were separated by 146-kb on the 8.1-Mb chromosome.

The regulatory mechanism of 2,4,6-trichlorophenol catabolic operon expression by HadR in ralstonia pickettii DTP0602

Ralstonia pickettii DTP0602 utilizes 2,4,6-trichlorophenol (2,4,6-TCP) as its sole source of carbon. The expression of catabolic pathway genes (hadA, hadB and hadC) for 2,4,6-TCP has been reported to be regulated by the LysR-type transcriptional regulator (LTTR) HadR. Generally, coinducers are recognized as being important for the function of LTTRs, and alteration of the LTTR-protection sequence and the degree of DNA bending are characteristic of LTTRs with or without a recognized coinducer. In this study, we describe the mechanism by which HadR regulates the expression of 2,4,6-TCP catabolic genes. The 2,4,6-TCP catabolic pathway genes in DTP0602 consist of two transcriptional units: hadX-hadA-hadB-hadC and monocistronic hadR. Purified HadR binds to the hadX promoter and HadR-DNA complex formation was induced in the presence of 16 types of substituted phenols, including chloro- and nitro-phenols and tribromo-phenol. In contrast with observations of other well-characterized LTTRs, the tested phenols showed no diversity of the bending angle of the HadR binding fragment. The expression of 2,4,6-TCP catabolic pathway genes, which are regulated by HadR, was not influenced by the DNA bending angle of HadR. Moreover, the transcription of hadX, hadA and hadB was induced in the presence of seven types of substituted phenols, whereas the other substituted phenols, which induced formation of the HadR-DNA complex, did not induce the transcription of hadX, hadA or hadB in vivo.

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.