Naoto Ogawa

Crystal Structure of a Full-length LysR-type Transcriptional Regulator, CbnR: Unusual Combination of Two Subunit Forms and Molecular Bases for Causing and Changing DNA Bend

The LysR-type transcriptional regulator (LTTR) proteins are one of the most common transcriptional regulators in prokaryotes. Here we report the crystal structure of CbnR, which is one of the LTTRs derived from Ralstonia eutropha NH9. This is the first crystal structure of a full-length LTTR. CbnR was found to form a homo-tetramer, which seems to be a biologically active form. Surprisingly, the tetramer can be regarded as a dimer of dimers, whereby each dimer is composed of two subunits in different conformations. In the CbnR tetramer, the DNA-binding domains are located at the V-shaped bottom of the main body of the tetramer, and seem to be suitable to interact with a long stretch of the promoter DNA, which is approximately 60bp. Interaction between the four DNA-binding domains and the two binding sites on the target DNA is likely to bend the target DNA along the V-shaped bottom of the CbnR tetramer. The relaxation of the bent DNA, which occurs upon inducer binding to CbnR, seems to be associated with a quaternary structure change of the tetramer.

The chlorocatechol degradative genes, tfdT-CDEF, of Burkholderia sp. strain NK8 are involved in chlorobenzoate degradation and induced by chlorobenzoates and chlorocatechols.

The modified-ortho pathway genes responsible for the degradation of chlorocatechols produced from 3- and 4-chlorobenzoate in Burkholderia sp. NK8 were cloned and analyzed. The five genes predicted to encode a LysR-type transcriptional regulator, chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase were designated tfdT, tfdC, tfdD, tfdE, and tfdF, respectively since they show the highest similarity to the corresponding genes of the chlorocatechol degradation gene cluster (tfdT-CDEF) of 2,4-dichlorophenoxyacetic acid degrading plasmid pJP4 from Ralstonia eutropha JMP134 (79-88% amino acid identity). TfdC of NK8 showed the highest activity against 3,5-dichlorocatechol in all kinds of chlorocatechols tested, which is a characteristic of TfdC of pJP4. By reporter gene (lacZ) analysis, tfdT of NK8 was shown to activate the transcription from the tfdC promoter. Unlike the regulators of other chlorocatechol degradation genes so far reported, 2-chlorobenzoate, 3-chlorobenzoate, 3-chlorocatechol and 4-chlorocatechol, were shown to act as effectors of TfdT.

Expression of 2-halobenzoate dioxygenase genes (cbdSABC) involved in the degradation of benzoate and 2-halobenzoate in Burkholderia sp. TH2

Burkholderia sp. TH2, isolated from soil, utilizes 2-chlorobenzoate (2CB) and benzoate (BA) as its sole source of carbon and energy. The genes for 2-halobenzoate dioxygenase (cbdABC) from Burkholderia sp. TH2 were cloned and sequenced. The predicted amino acid sequences of all the gene products are highly similar to the cbd gene products of Pseudomonas sp. 2CBS. Disruption of the promoter region of cbdA resulted in loss of growth on 2CB and BA, indicating that these genes are involved in the growth of TH2 on these substrates. Expression of the cbd genes was analyzed by transcriptional fusion assay. The cbdS gene, a possible araC/xylS-type transcriptional regulatory gene, was shown to positively regulate the expression of cbdA. In addition, the effectors of CbdS were shown to be 2CB, 2-bromobenzoate, o-toluate (2-methylbenzoate), 2-iodobenzoate, and BA. Primer extension analysis showed that the cbdA mRNA started at two positions, 14 and 15 nucleotides upstream from the cbdA start codon, ATG. A pair of direct repeats, identical to that of the Pm promoter of the TOL plasmid, was found upstream of -35 hexamer of the cbdA promoter.

Differential Expression of Two Catechol 1,2-Dioxygenases in Burkholderia sp. Strain TH2

Burkholderia sp. strain TH2, a 2-chlorobenzoate (2CB)-degrading bacterium, metabolizes benzoate (BA) and 2CB via catechol. Two different gene clusters for the catechol ortho-cleavage pathway (cat1 and cat2) were cloned from TH2 and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that while both catechol dioxygenases (CatA1 and CatA2) were produced in BA-grown cells, CatA1 was undetectable when strain TH2 was grown on 2CB or cis,cis-muconate (CCM), an intermediate of catechol degradation. However, production of CatA1 during growth on 2CB or CCM was observed when cat2 genes were disrupted. The difference in the production of CatA1 and CatA2 was apparently due to a difference in inducer recognition by the regulators of the gene clusters. The inducer of CatA1 was found to be BA, not 2CB, by using a 2-halobenzoate dioxygenase gene (cbd) disruptant, which is incapable of transforming (chloro)benzoate. It was also found that CCM or its metabolite acts as an inducer for CatA2. When cat2 genes were disrupted, the growth rate in 2CB culture was reduced while that in BA culture was not. These results suggest that although cat2 genes are not indispensable for growth of TH2 on 2CB, they are advantageous.

The chlorobenzoate dioxygenase genes of Burkholderia sp. strain NK8 involved in the catabolism of chlorobenzoates.

Burkholderia sp. NK8 grows abundantly on 3-chlorobenzoate (3CB),4-chlorobenzoate (4CB) and benzoate. The genes encoding the oxidation of (chloro)benzoates (cbeABCD) and catechol (catA, catBC), the LysR-type regulatory gene cbeR and the gene cbeE with unknown function, all of which form a single cluster in NK8, were cloned and analysed. The protein sequence of chlorobenzoate 1,2-dioxygenase (CbeABC) is 50-65% identical to the benzoate dioxygenase (BenABC) of Acinetobacter sp. ADP1, toluate dioxygenase (XylXYZ) of the TOL plasmid pWW0 and 2-halobenzoate dioxygenase (CbdABC) of Burkholderia cepacia 2CBS. Disruption of the cbeA gene resulted in the simultaneous loss of the ability to grow on benzoate and monochlorobenzoates, indicating the involvement of the cbeABCD genes in the degradation of these aromatics. The cbeABCD genes are preceded by catA, the gene for catechol dioxygenase. lacZ transcriptional fusion studies in Pseudomonas putida showed that catA and cbeA are co-expressed under the positive control of cbeR, a LysR-type transcriptional regulatory gene. The cbeA::lacZ transcriptional fusion studies showed that the inducers of the genes are 3CB, 4CB, benzoate and probably cis,cis-muconate. On the other hand, 2-chlorobenzoate (2CB) did not activate the expression of the genes. The chlorobenzoate dioxygenase was able to transform 2CB, 3CB, 4CB and benzoate at considerable rates. 2CB yielded both catechol and 3-chlorocatechol (3CC), and 3CB gave rise to 4-chlorocatechol and 3CC as the major and minor intermediate products, respectively, indicating that the NK8 dioxygenase lacks absolute regiospecificity. The absence of growth of NK8 on 2CB, despite its considerable degradation activity against 2CB, is apparently due to the inability of CbeR to recognize 2CB as an inducer of the expression of the cbe genes.

An improved method to extract RNA from soil with efficient removal of humic acids.

Aims:  To remove humic substances from RNA extracted from soil for the study of bacterial gene expression in soil.

Detection of bphAa Gene Expression of Rhodococcus sp. Strain RHA1 in Soil Using a New Method of RNA Preparation from Soil

To understand the response of soil bacteria to the surrounding environment, it is necessary to examine the gene expression profiles of the bacteria in the soil. For this purpose, we developed a new method of extracting RNA from soil reproducibly. Using this new method, we extracted RNA from a field soil, which was sterilized and inoculated with Rhodococcus sp. strain RHA1, a biphenyl degrader isolated from γ-hexachlorocyclohexane-contaminated soil. Data from agarose gel electrophoresis indicated that the extracted RNA was purified properly. This new method can be applied easily in the preparation of large amounts of RNA. Real-time reverse transcription-polymerase chain reaction (RT-PCR) experiments performed by the TaqMan method suggested that the bphAa gene in this strain, which is involved in the degradation of biphenyl, was induced in the biphenyl amended soil.

Molecular Breeding of Transgenic Rice Plants Expressing a Bacterial Chlorocatechol Dioxygenase Gene

ABSTRACT The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.

Enhanced biofilm formation and 3-chlorobenzoate degrading activity by the bacterial consortium of Burkholderia sp. NK8 and Pseudomonas aeruginosa PAO1

Aims:  To characterize biofilm formation of a chlorobenzoates (CBs) degrading bacterium, Burkholderia sp. NK8, with another bacterial species, and the biodegradation activity against CBs in the mixed‐species biofilm.

New analytical method for the determination of styrene oligomers formed from polystyrene decomposition and its application at the coastlines of the North-West Pacific ocean.

The pollution caused by plastic debris is an environmental problem with increasing concern in the oceans. Among the plastic polymers, polystyrene (PS) is one of the most problematic plastics due to the direct public health risk associated with their dispersion, as well as the numerous adverse environmental impacts which arise both directly from the plastics and from their degradation products. Little is known about their potential distribution characteristics throughout the oceans. For the first time, we report here on the regional distribution of styrene monomer (SM), styrene dimers (SD; 2,4-diphenyl-1-butene, SD1; 1,3-diphenyl propane, SD2), and styrene trimer (2,4,6-triphenyl-1-hexene: ST1), as products of PS decomposition determined from samples of sand and seawater from the shorelines of the North-West Pacific ocean. In order to quantitatively determine SM, SD (=SD1+SD2), and ST1, a new analytical method was developed. The detection limit was 3.3 μg L(-1), based on a signal-to-noise ratio of three, which was well-suited to quantify levels of SM, SD, and ST1 in samples. Surprisingly, the concentrations of SM, SD, and ST1 in sand samples from the shorelines were consistently greater than those in seawater samples from the same location. The results of this study suggest that SM, SD, and ST1 can be widely dispersed throughout the North-West Pacific oceans.