Hisashi Takeda

Characterization of Transcriptional Regulatory Genes for Biphenyl Degradation in Rhodococcus sp. Strain RHA1

Transcription of the bphA1A2A3A4C1B genes, which are responsible for the conversion of biphenyl and polychlorinated biphenyl to the meta-cleavage products in Rhodococcus sp. strain RHA1, was examined. The bphA1 promoter (P(bphA1)) was identified and was shown to promote transcription induction by biphenyl and ethylbenzene. An 8.8-kb HindIII fragment that promotes transcription induction of P(bphA1) in Rhodococcus erythropolis IAM1399 was isolated from the region downstream of bphB by using a reporter plasmid containing P(bphA1). Analysis of the nucleotide sequence of this fragment revealed a set of putative two-component regulatory system genes, which were designated bphS and bphT. Deletion analysis of the 8.8-kb HindIII fragment indicated that bphT is responsible for the basal activation of P(bphA1) and that both bphS and bphT are required for the elevated basal activation of and transcriptional induction by biphenyl of P(bphA1). These results support the notion that bphS and bphT encode a sensor kinase and a response regulator, respectively, of a two-component regulatory system. The bphS and bphT genes promote transcriptional induction by a variety of aromatic compounds, including biphenyl, benzene, alkylbenzenes, and chlorinated benzenes. A promoter activity assay and reverse transcription (RT)-PCR analysis revealed a weak constitutive promoter in the adjacent region upstream of bphS. RT-PCR analysis indicated that there is induced transcription of bphA1 through bphT, in which P(bphA1) is thought to take part. An insertionally inactivated bphS mutant, SDR1, did not grow on biphenyl. Growth was restored by introduction of an intact bphS gene into SDR1. These results indicate that at least bphS is indispensably responsible for the growth of RHA1 on biphenyl.

Biphenyl-inducible promoters in a polychlorinated biphenyl-degrading bacterium, Rhodococcus sp. RHA1.

Five transcriptional promoters of biphenyl-degradation genes in Rhodococcus sp. RHA1 were characterized. We newly identified the etbA4 promoter region, which was located adjacent upstream from a ferredoxin reductase gene, etbA4 and a dihydrodiol dehydrogenase gene, bphB2. The etbA4 promoter activity was determined in RHA1 using a promoter probe vector with a luxAB luciferase reporter gene, and was induced by a variety of aromatic compounds as well as the bphA1, ebdA1, etbA1, and etbD1 promoters. All these promoters were induced by aromatic compounds in a closely related heterologous host, R. erythropolis IAM1399 in the presence of RHA1 bphST genes, suggesting that these five promoters are under the control of bphST-coding two-component regulatory system. Sequence comparison of the bphA1 promoter with the ebdA1 and etbA1 promoters, whose transcription starts were determined by primer extension analysis, revealed a consensus sequence centering 42-bp upstream from the transcription start. This consensus was also conserved in the etbA4 and etbD1 promoters, and deletions of the bphA1 promoter affecting the consensus impaired inducible promoter activity. These results suggest that this consensus plays a role in transcription induction and/or the promotion of biphenyl degradation genes in RHA1.

Characterization of Two Biphenyl Dioxygenases for Biphenyl/PCB Degradation in A PCB Degrader, Rhodococcus sp. Strain RHA1

Rhodococcus sp. RHA1 induces two biphenyl dioxygenases, the BphA and EtbA/EbdA dioxygenases, during growth on biphenyl. Their subunit genes were expressed in R. erythropolis IAM1399 to investigate the involvement of each subunit gene in their activity and their substrate preferences. The recombinant expressing ebdA1A2A3etbA4 and that expressing bphA1A2A3A4 exhibited 4-chlorobiphenyl (4-CB) transformation activity, suggesting that these gene sets are responsible for the EtbA/EbdA and BphA dioxygenases respectively. When bphA4 and etbA4 were swapped to construct the recombinants expressing ebdA1A2A3bphA4 and bphA1A2A3etbA4 respectively, compatibility between BphA4 and EtbA4 was suggested by their 4-CB transformation activities. When bphA3 and ebdA3 were swapped, incompatibility between BphA3 and EbdA3 was suggested. BphA and EtbA/EbdA dioxygenases exhibited the highest transformation activity toward biphenyl and naphthalene respectively, and also attacked dibenzofuran and dibenzo-p-dioxin. The wide substrate preference of EtbA/EbdA dioxygenase suggested that it plays a more important role in polychlorinated biphenyl (PCB) degradation than does BphA dioxygenase.

Heat stable ssDNA/RNA-binding activity of a wheat cold shock domain protein

The cold‐induced wheat WCSP1 protein belongs to the cold shock domain (CSD) protein family. In prokaryotes and eukaryotes, the CSD functions as a nucleic acid‐binding domain. Here, we demonstrated that purified recombinant WCSP1 is boiling soluble and binds ss/dsDNA and mRNA. Furthermore, boiled‐WCSP1 retained its characteristic nucleic acid‐binding activity. A WCSP1 deletion mutant, containing only a CSD, lost ssDNA/RNA‐binding activity; while a mutant containing the CSD and the first glycine‐rich region (GR) displayed the activity. These data indicated that the first GR of WCSP1 is necessary for the binding activity but is not for the heat stability of the protein.

Dual Two-Component Regulatory Systems Are Involved in Aromatic Compound Degradation in a Polychlorinated-Biphenyl Degrader, Rhodococcus jostii RHA1

A Gram-positive polychlorinated-biphenyl (PCB) degrader, Rhodococcus jostii RHA1, degrades PCBs by cometabolism with biphenyl. A two-component BphS1T1 system encoded by bphS1 and bphT1 (formerly bphS and bphT) is responsible for the transcription induction of the five gene clusters, bphAaAbAcAdC1B1, etbAa1Ab1CbphD1, etbAa2Ab2AcD2, etbAdbphB2, and etbD1, which constitute multiple enzyme systems for biphenyl/PCB degradation. The bphS2 and bphT2 genes, which encode BphS2 and BphT2, virtually identical to BphS1 (92%) and BphT1 (97%), respectively, were characterized. BphS2T2 induced the activation of the bphAa promoter in a host, Rhodococcus erythropolis IAM1399, in the presence of a variety of aromatics, including benzene, toluene, ethylbenzene, xylenes, isopropylbenzene, and chlorinated benzenes, as effectively as BphS1T1. The substrate spectrum of BphS2T2 was the same as that of BphS1T1, except for biphenyl, which is a substrate only for BphS1T1. BphS2T2 activated transcription from the five promoters of biphenyl/PCB degradation enzyme gene clusters as effectively as BphS1T1. The targeted disruptions of the bphS1, bphS2, bphT1, and bphT2 genes indicated that all these genes are involved in the growth of RHA1 on aromatic compounds. The hybrid system with bphS1 and bphT2 and that with bphS2 and bphT1 were constructed, and both systems conducted induced activation of the bphAa promoter, indicating cross-communication. These results indicated that RHA1 employs not only multiple enzyme systems, but also dual regulatory systems for biphenyl/PCB degradation. Comparison of the sequences, including bphS2T2, with the bphS1T1-containing sequences and the corresponding sequences in other rhodococcal degraders suggests that bphS2T2 might have originated from bphS1T1.

Crystallization of the terminal oxygenase component of biphenyl dioxygenase derived from Rhodococcus sp. strain RHA1.

The terminal oxygenase component of the biphenyl dioxygenase (BphA1A2 complex) was over-expressed with a novel over expression system in recombinant Rhodococcus strain and purified. The purified enzyme has been crystallized by the hanging drop vapor diffusion method and subjected to X-ray diffraction analysis. The crystals belong to the tetragonal system in the space group P4(1)2(1)2 or P4(3)2(1)2 and diffract to better than 2.2A resolution.

Isolation and structure elucidation of new phthalide and phthalane derivatives, isolated as antimicrobial agents from Emericella sp. IFM57991.

Three new phthalide derivatives, emefuranones A1, A2 and B (1–3); six new phthalane derivatives, emefuran A, B1, B2, C1, C2 and D (4–9); three new farnesylated phthalide derivatives, farnesylemefuranones A–C (10–12); xylarinol C (13); and emericelloxide (14), along with four known compounds (dustanin, sorbicillin, aspergillodiol and xylarinol A), were isolated from the culture extracts of Emericella sp. IFM57991. Structures of 1–14 were elucidated on the basis of spectroscopic analysis and chemical evidence. Compounds 4–7 and 13 showed moderate antibacterial activities against Bacillus subtilis.

Isolation of growth inhibitors of the snow rot pathogen Pythium iwayamai from an arctic strain of Trichoderma polysporum

Growth inhibitors were isolated from an arctic strain of Trichoderma polysporum, and the structures were elucidated and the in vitro inhibitory effects of these compounds against Pythium iwayamai were investigated. Eleven compounds were isolated; four showed a concentration-dependent growth-inhibitory effect against P. iwayamai. None of these compounds have been reported previously as substances with antimicrobial activity against P. iwayamai. One of these four compounds inhibited the growth of the pathogen at 33 μg ml−1 concentration during a 15-day incubation at 20 °C. This effect was comparable to that of chloroneb (1: 1,4-dichloro-2,5-dimethoxybenzene), a fungicide with activity against P. iwayamai. Thus, the results of the present study show that the arctic strain of T. polysporum can be an effective source of antibiotics with activity against the snow rot pathogen, P. iwayamai.

11-Hydroxylation of Protoberberine by the Novel Berberine-Utilizing Aerobic Bacterium Sphingobium sp. Strain BD3100.

Protoberberine alkaloids, including berberine, palmatine, and berberrubine, are produced by medicinal plants and are known to have various pharmacological effects. We isolated two berberine-utilizing bacteria, Sphingobium sp. strain BD3100 and Rhodococcus sp. strain BD7100, from soil collected at a natural medicine factory. BD3100 had the unique ability to utilize berberine or palmatine as the sole carbon and energy source. BD3100 produced demethyleneberberine in berberine-supplemented medium. In a resting-cell incubation with berberine, BD3100 produced 11-hydroxyberberine; the structure of 11-hydroxyberberine was determined by detailed analysis of NMR and MS spectroscopic data. α-Naphthoflavone, miconazole, and ketoconazole, which are known inhibitors of cytochrome P450, interfered with BD3100 metabolism of berberine in resting cells. Inhibition by miconazole led to the production of a new compound, 11-hydroxydemethyleneberberine. In a resting-cell incubation with palmatine, BD3100 generated 11-hydroxypalmatine. This work represents the first report of the isolation and characterization of novel berberine-utilizing aerobic bacteria for the production of 11-hydroxylation derivatives of berberine and palmatine.

Asnovolins A–G, Spiromeroterpenoids Isolated from the Fungus Aspergillus novofumigatus, and Suppression of Fibronectin Expression by Asnovolin E

Seven novel spiromeroterpenoids, asnovolins A-G (1-7), one of which was shown to suppress fibronectin expression, were isolated from Aspergillus novofumigatus CBS117520 along with a known compound, novofumigatonin (8). The structures of asnovolins A-G were elucidated using MS and 2D-NMR data. Asnovolin E (5) suppressed fibronectin expression by normal human neonatal dermal fibroblast cells.