Takashi Hatta

The bphDEF meta-cleavage pathway genes involved in biphenyl/polychlorinated biphenyl degradation are located on a linear plasmid and separated from the initial bphACB genes in Rhodococcus sp. strain RHA1

The bphACB genes responsible for the initial oxidation of the aromatic ring of biphenyl/polychlorinated biphenyls (PCB) to meta-cleavage product in Rhodococcus sp. RHA1 have been characterized. We cloned the 6.1 kb EcoRI fragment containing another extradiol dioxygenase gene (etbC) which was induced during the growth on ethylbenzene. The bphD, bphE and bphF encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase, 2-hydroxypenta-2,4-dienoate hydratase and 4-hydroxy-2-oxovalerate aldolase, respectively, were found downstream of etbC. The deduced amino acid (aa) sequence of RHA1 bphD and bphE had 27-33% and 32-38% identity, respectively, with those of the corresponding genes in Pseudomonas. BphE and BphF are closely related to the corresponding homoprotocatechuate meta-cleavage pathway enzymes of Escherichia coli C. The bphD and bphF were expressed in E. coli and the BphD activity was detected. The etbCphDEF genes were transcribed in biphenyl and ethylbenzene growing cells. Pulsed field gel electrophoresis (PFGE) analysis indicated that RHA1 contains three large linear plasmids. Southern blot analysis indicated that the meta-cleavage pathway for biphenyl/PCB catabolism in RHA1 is directed by the 390 kb plasmid borne bphDEF genes located separately from bphACB gene cluster on the 1100 kb plasmid.

PcpA, which is involved in the degradation of pentachlorophenol in Sphingomonas chlorophenolica ATCC39723, is a novel type of ring‐cleavage dioxygenase

The pentachlorophenol (PCP) mineralizing bacterium Sphingomonas chlorophenolica ATCC39723 degrades PCP via 2,6‐dichlorohydroquinone (2,6‐DCHQ). The pathway converting PCP to 2,6‐DCHQ has been established previously; however, the pathway beyond 2,6‐DCHQ is not clear, although it has been suggested that a PcpA plays a role in 2,6‐DCHQ conversion. In this study, PcpA expressed in Escherichia coli was purified to homogeneity and shown to have novel ring‐cleavage dioxygenase activity in conjunction with hydroquinone derivatives, and converting 2,6‐DCHQ to 2‐chloromaleylacetate.

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 .

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.

Prophenol Oxidase A3 in Drosophila melanogaster: Activation and the PCR-Based cDNA Sequence

Phenol oxidase exists in Drosophila hemolymph as a prophenol oxidase, A1 and A3, that is activated in vivo with a native activating system, AMM-1, by limited proteolysis with time. The polypeptide in purified prophenol oxidase A3 has a molecular weight of approximately 77,000 Da. A PCR-based cDNA sequence coding A3 has 2501 bp encoding an open reading frame of 682 amino acid residues. The potential copper-binding sites, from Trp-196 to Tyr-245, and from Asn-366 to Phe-421, are highly homologous to the corresponding sites in other invertebrates. The availability of prophenol oxidase cDNA should be useful in revealing the biochemical differences between A1 and A3 isoforms in Drosophila melanogaster that are refractory or unable to activate prophenol oxidase.

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 changes in congener selectivity during PCB degradation by Burkholderia sp. strain TSN101 with increasing concentrations of PCB and characterization of the bphBCD genes and gene products

Abstract We isolated and characterized a gram-negative bacterium, Burkholderia sp. strain TSN101, that can degrade polychlorinated biphenyls (PCBs) at concentrations as high as 150 μg Kaneclor 300/ml, a PCB mixture equivalent to Aroclor 1242. Growing cells of strain TSN101 degraded most of the tri- and tetrachlorobiphenyls in medium containing 25 μg Kaneclor 300/ml. Using PCB concentrations of 50–150 μg of Kaneclor 300/ml, the congener selectivity pattern was different and the pattern of chlorine substitution strongly affected degradation of some congeners. At 25 μg Kaneclor 300/ml, strain TSN101 degraded di- and trichlorinated congeners with chlorine substitutions at both the ortho and the para positions. At higher concentrations of Kaneclor 300, di- and trichlorobiphenyls with ortho substituents in both phenyl rings were not degraded well. Trichlorobiphenyls with para and meta substitutents were degraded equally well at all concentrations studied. The ability of strain TSN101 to degrade ortho and para-substituted congeners was confirmed using a defined PCB mixture with chlorine substituents at 2′- and 4′-positions. A 5-kb DNA fragment containing the bphBCD genes was cloned and sequenced. Comparison of the deduced amino acid sequences of these genes with related proteins indicated 99 and 98% sequence similarity to the BphB and BphD of Comamonas testosteroni strain B-356, respectively. The bphC gene product showed 74% sequence similarity to the BphC of Burkholderia cepacia strain LB400 and exhibited a narrow substrate specificity with strong affinity for 2,3-dihydroxybiphenyl. A bphC-disrupted mutant of Burkholderia sp. strain TSN101, constructed by gene replacement, lost the ability to utilize biphenyl, thus supporting the role of the cloned bph gene in biphenyl metabolism.

Complete Genome Sequence of the Thermophilic Polychlorinated Biphenyl Degrader Geobacillus sp. Strain JF8 (NBRC 109937).

ABSTRACT Geobacillus sp. strain JF8 (NBRC 109937) utilizes biphenyl and naphthalene as sole carbon sources and degrades polychlorinated biphenyl (PCB) at 60°C. Here, we report the complete nucleotide sequence of the JF8 genome (a 3,446,630-bp chromosome and a 39,678-bp plasmid). JF8 has the smallest genome among the known PCB degraders.

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.